The body fluids of marine teleost (bony) fish—such as these coral reef fish—are far more dilute than the seawater in which they are swimming. Such fish have blood osmotic pres- sures of about 300–500 milliosmolar (mOsm), whereas seawater has an osmotic pressure of approximately 1000 mOsm. From the viewpoint of fluid concentration, marine teleost fish are packets of low-salinity fluids cruising about within a high-salinity environment!

Because the body fluids of a marine teleost fish are not at equilibrium with the seawater surrounding the fish, passive processes occur that tend to alter the composition of the body fluids. Water tends to move out of the body fluids into the surrounding seawater by osmosis because the body fluids have a lower osmotic pressure than seawater. Conversely, several inorganic ions diffuse inward from the concentrated seawater. Both the outward osmosis of water and the inward diffusion of ions tend to concentrate the fish’s body fluids. Recognizing these processes, we see that marine teleost fish must steadily expend energy to maintain their body fluids out of equilibrium with the seawater in which they swim.

When we reflect on the questions that are raised by the body-fluid composition of marine teleost fish, we quickly recognize that the questions of mechanism and origin stressed in Chapter 1

These ocean fish (powder blue surgeonfish, Acanthurus leucosternum) expend en- ergy to keep their body fluids more dilute than seawater Major questions raised are why they do so and what mechanisms they employ.

742 Chapter 28

are both important. How do the fish keep their body fluids more dilute than seawater, and why do they do so? These same two ques- tions—which are the central questions of mechanistic physiology and evolutionary physiology—arise in the study of the water–salt physiology of all animals.

In terms of their water–salt physiology, animals have been versatile in adapting to an astounding range of environments on Earth—not just seawater and ordinary freshwater, but also salt lakes far more concentrated than seawater, glacial ponds almost as dilute as distilled water, estuaries with highly variable salinity, and terrestrial environments ranging from rainforests to extreme deserts. Each type of habitat poses distinct challenges, and animals of diverse types that live in a particular habitat often have converged on similar mechanisms for meeting the challenges. For these reasons, the detailed study of animal water–salt physiology is logically organized around habitats. We take that approach in this chapter. We start by focusing on freshwater habitats, in part because we have already emphasized them in Chapter 5, and in part because the study of freshwater fish will help set the stage for a better understanding of ocean fish.

Animals in Freshwater

The animals living today in freshwater are descended from ocean- living ancestors: The major animal phyla originated in the oceans and later invaded all other habitats. Seawater was probably somewhat different in its total salinity and salt composition in the early eras of animal evolution than it is today. Nonetheless, when the animal phyla invaded freshwater from the oceans, there can be no doubt that they encountered a drastic reduction in the concentration of their surroundings. The osmotic pressure of freshwater is typically less than 1% as high as that of seawater today, and the major ions in freshwater are very dilute compared with their concentrations in seawater (see Table 27.1).

All freshwater animals regulate their blood1 osmotic pressures at levels hyperosmotic to freshwater and are therefore classified as

1 In this book, we always use the term blood to refer to the fluid that is circulated within the circulatory system, although some authors use the term hemolymph to refer to that fluid in invertebrates that have open circulatory systems (see page 695).

hyperosmotic regulators. As TAblE 28.1 shows, the blood osmotic pressures of various types of freshwater animals span an order of magnitude, but even freshwater mussels, which are among the most dilute animals on Earth, have blood that is substantially more concentrated than freshwater; body fluids as dilute as freshwater seem to be incompatible with life. The solutes in the blood plasma of freshwater animals are mainly inorganic ions; Na+ and Cl– dominate. As Table 28.1 shows, each of the individual inorganic ions in the blood plasma of freshwater animals is—in almost all cases—substantially more concentrated in the blood than in freshwater.

Passive water and ion exchanges: Freshwater animals tend to gain water by osmosis and lose major ions by diffusion

Being hyperosmotic to their surroundings, freshwater animals tend to gain water continuously by osmosis, and this water gain tends to dilute their body fluids. The relatively high concentrations of ions in their blood suggest that the net diffusion of ions tends to be from their blood into the ambient water. The analysis of ion diffusion is actually more complex, however, because—as discussed in Chapter 5—ion diffusion depends on electrical gradients as well as concentration gradients. When all the complexity is taken into account, nevertheless (see pages 110–111), the direction of diffusion of the major ions—such as Na+ and Cl–—is as the concentration gradients suggest: from the blood into the environmental water. This loss of major ions by dif- fusion tends, like the osmotic water gain, to dilute the body fluids of a freshwater animal as shown at the top in FigurE 28.1.

In a broad sense, we expect a freshwater animal’s energy costs for osmotic and ionic regulation to depend directly on the animal’s rates of passive water gain and passive ion loss. The more rapidly water is taken up by osmosis, and the more rapidly ions are lost by diffusion, the more rapidly an animal will need to expend energy to counteract these processes so as to maintain a normal blood composition. Three factors determine the rates of passive exchange of water and ions: (1) the magnitudes of the osmotic and ionic gra- dients between the blood and ambient water, (2) the permeability of an animal’s outer body covering to water and ions, and (3) the surface area across which exchange is occurring. The first two of these factors deserve further discussion.

Source: Hill and Wyse 1989; river water data from Table 27.1.
a The frog has recently been renamed Pelophylax esculentus.

(A) Problems of passive water and salt exchange faced by freshwater animals

FigurE 28.1 Water–salt relations in a fresh- water animal (A) A freshwater animal, such as a crayfish, faces challenges because of passive water and salt exchange.The numbers are gener- alized, approximate values for the osmotic pressure and Na+ concentration found in the blood of a crayfish and the ambient water.Values for a spe- cific species of crayfish under specific study condi- tions are given in Table 28.1. (B) A summary of all the major processes of water and salt exchange, including the energy-requiring processes the animal uses to maintain water–salt balance.The antennal glands, or green glands, which function as the kidneys of a crayfish, open at the bases of the second antennae.

cause their blood is less concentrated than that of their marine progenitors. In the crayfish in Table 28.1, for example, the osmotic difference between the blood and the surrounding water is about 440 mOsm.

The evolution of more-dilute blood when animals invaded freshwater was probably an adaptation to reduce the energy costs of living in freshwater. More-dilute blood means smaller osmotic and ionic gradients between the blood and freshwater, and thus lower rates of water gain and ion loss by osmosis and diffusion.

PErMEAbiliTiES The permeability of the integument2 of a freshwater animal to water and ions is in general relatively low. Freshwater crayfish, for example, are no more than 10% as permeable to water, Na+, and Cl– as marine decapod crustaceans of similar body size. The low permeabilities evolved by freshwater ani- mals are important in reducing their rates of passive water and ion exchange and thus in reducing their energy costs of maintaining a normal blood composition. For a freshwater

animal (or any other animal that maintains a difference in com- position between its blood and the ambient water), a low integu- mentary permeability is analogous to an insulatory pelage in an Arctic mammal; the low permeability slows the processes that tend to bring the blood and ambient water to equilibrium, just as pelage insulation slows heat losses that tend to cool an Arctic mammal to ambient temperature.

If freshwater animals did not need to breathe, they might cover themselves entirely in an integument of extremely low permeability to water and ions. However, they do need to breathe, and there seems to be no way to make gills that are both highly permeable to O2 and poorly permeable to H2O and inorganic ions. Thus, just as the gills of freshwater animals provide a “window” for O2 to enter the body, they provide a window for water to enter by osmosis and for ions to leave by diffusion. In fact, the very attributes of gills that are virtues

2 Integument is a general term for the outer body covering. For example, the integument of a vertebrate is its skin, and the integument of an arthropod is its exoskeleton (cuticle) or shell.

Salt loss by diffusion

Water gain by osmosis

Carapace

Antennal gland (green gland), responsible for urine formation

Water and Salt Physiology of Animals in Their Environments 743

The gills, which are covered by the carapace and not visible externally, project into the branchial chambers, through which water is pumped.

A freshwater animal tends to gain water and lose salts, especially across its gills.

(B) Summary of all water and salt exchanges

Salt loss by diffusion

Water gain by osmosis

Blood

̃ 400 mOsm + ̃ 200 mM Na

Ambient water

̃ 3 mOsm + ̃ 0.4 mM Na

Copious, dilute urine Salts and water in food

Most types of freshwater animals have far less concentrated body fluids than their marine relatives. The decapod crustaceans (e.g., crayfish, crabs, and lob- sters) illustrate this general pattern. Although most marine deca- pods are essentially isosmotic to seawater (about 1000 mOsm), most freshwater decapods have blood osmotic pressures of 500 mOsm or less (e.g., ~440 mOsm in the crayfish in Table 28.1). Similarly, although marine molluscs are approximately isosmotic to seawater, freshwater molluscs have far lower blood osmotic pressures (e.g., ~40–80 mOsm in those in Table 28.1). The lower blood concentra- tions seen in freshwater animals result in smaller osmotic and ionic gradients between their blood and the freshwater environment than would otherwise be the case. For example, if decapod crusta- ceans and molluscs had retained their ancient blood concentrations when they invaded freshwater, the osmotic difference between their blood and freshwater would be almost 1000 mOsm. The actual osmotic difference between the blood and the surrounding water in freshwater decapod crustaceans and molluscs is far lower be-

hysiology 4E iates 12-23-15

Salts and water in feces

Active absorption of Na+ and Cl–

OSMOTiC ANd iONiC grAdiENTS

P

c

744 Chapter 28

Source: Hill and Wyse 1989.
aThe osmotic U/P ratio is the osmotic pressure of the urine divided by the osmotic pressure of

the blood plasma.
bThe Na+ U/P ratio is the urine Na+ concentration divided by the plasma Na+ concentration.

a measure of the rate of osmotic water influx. The daily osmotic water influx of a goldfish or frog is therefore equal to one-third of its body weight!4

The urine of freshwater animals, in addition to being produced in abun- dance, is typically markedly hyposmot- ic to their blood plasma and contains much lower concentrations of Na+ and Cl– than the plasma. That is, the U/P ratios (urine:plasma ratios) for osmotic pressure, Na+, and Cl– are far less than 1 in these animals (see Table 28.2). Recall from Figure 27.7 that when the osmotic U/P ratio is less than 1, urine production tends to raise the plasma osmotic pres- sure. Similarly, when the U/P ratio for an ion is less than 1, urine production tends to raise the plasma concentration of that ion. Typically, therefore, the kidneys of a

for O2 uptake—high permeability and large surface area—are negatives for water–salt balance. A common pattern in freshwater animals is for little osmosis and diffusion to occur across the general integument—because the integument is poorly permeable to water and ions—and for most osmosis and diffusion to occur across the gills (plus possibly a few other localized body surfaces3).

The importance of the gills as windows for passive water and ion exchange has an interesting and significant implication: Differ- ences in whole-body permeability to water and salts among related freshwater animals are sometimes secondary effects of differences in their metabolic intensities and demands for O2. Species with high O2 demands often have gill systems that are particularly well suited to rapid inward rates of O2 diffusion. As a corollary, their gill systems also permit particularly rapid rates of water uptake by osmosis and ion loss by diffusion. In these cases, high rates of water–salt exchange are consequences of the evolution of high metabolic intensity.

most types of freshwater animals share similar regulatory mechanisms

Most types of freshwater animals share a fundamentally similar suite of mechanisms for osmotic–ionic regulation. This suite of mechanisms is found in such phylogenetically diverse groups as freshwater teleost fish, lampreys, frogs, toads, soft-shelled turtles, freshwater mussels, crayfish, earthworms, leeches, and mosquito larvae.

urine As we have seen, freshwater animals are faced with a continuous influx of excess water by osmosis. They void this excess water by making a copious (abundant) urine. A goldfish or frog, for example, might excrete urine equivalent to one-third of its body weight per day (table 28.2). Because urine production balances osmotic water gain, the rate of urinary water excretion provides

3 The membranes of the buccal and opercular cavities in fish, for example, are relatively permeable compared with most body surfaces and are important sites of passive water–salt exchange. Little water or ion exchange occurs across an adult fish’s outer skin.

freshwater animal not only solve the animal’s volume-regulation problem by voiding the animal’s excess volume of water, but also aid osmotic and ionic regulation by helping to maintain a high osmotic pressure and high ion concentrations in the blood.

Whereas the urine of freshwater animals is generally copious and dilute, an important concept to keep in mind is that kidneys are regulatory organs: They characteristically adjust their function in ways that help to maintain stability of volume and composition in the body fluids. Thus the exact volume and composition of the urine vary with circumstances. For example, if a freshwater animal experiences an increase in the rate at which it takes in water by osmosis, its kidneys ordinarily increase their rate of urine production.

Although freshwater animals typically limit the concentrations of Na+ and Cl– in their urine to low levels, some loss of these ions in the urine is inevitable. This urinary loss of ions can pose a threat to the integrity of the body fluids when Na+ and Cl– are in short supply. The rate of loss of ions in the urine depends in part on the rate of urine production, and therefore on the rate of osmotic water flux into an animal. Any factor that increases an animal’s rate of osmotic water influx tends to increase the animal’s rate of ion loss. We see, therefore, that volume regulation and ionic regulation are basically at conflict with each other in freshwater animals.

active ion uptake in Freshwater animals in general

An important way that freshwater animals replace lost Na+ and Cl– is that they actively transport both ions into their blood directly from the pond or river water in which they live. We have just seen that freshwater animals lose ions in their urine. Earlier we saw that they also lose ions by direct outward diffusion across their per- meable body surfaces. The ions lost in these two ways need to be replaced. Freshwater animals do this by taking up Na+ and Cl– by active transport from their ambient water. Other ions may also be transported inward in this way. Freshwater fish, for example, take up Ca2+ from the ambient water by active transport.

4 If a 70-kg person had a similar weight-specific rate of water uptake, he or she would gain 23 L (6 gallons) of water per day and thus would need to excrete 23 L of urine per day.

The capacities of most freshwater animals for active uptake of Na+ and Cl– are remarkable. For example, some crayfish, fish, and frogs—which have Na+ and Cl– concentrations of 100–200 mM in their blood plasma—can actively take up Na+ and Cl– in net fashion from ambient waters as dilute as 0.01 mM (four orders of magni- tude more dilute than their blood)! The site of active ion uptake is usually the gills or the general integument. In teleost (bony) fish and decapod crustaceans (e.g., crayfish), the site of uptake is the gill epithelium. In frogs, active ion uptake occurs across the gills when the animals are tadpoles but across the skin when they are adults.5 Active ion uptake also occurs across the general integument in leeches and aquatic oligochaete worms.

The cellular-molecular mechanisms of active Na+ and Cl– uptake by freshwater animals are featured as one of the focal examples in Chapter 5. Although details may vary, the following key points are believed to apply to all or most groups of freshwater animals:

The active uptake of ions from the ambient water requires ATP. Thus active ion uptake places demands on an animal’s energy resources.

The mechanisms that pump Na+ and Cl– from the ambient water into the blood are typically different and independent from each other.

The Cl– pump typically exchanges bicarbonate ions (HCO3–) for Cl– ions, in this way remaining electroneutral (FigurE 28.2).

The Na+ pump typically exchanges protons (H+) for Na+ ions (or possibly exchanges ammonium ions, NH4+,
for Na+ in some groups of animals), thereby remaining electroneutral (see Figure 28.2).

The HCO3– and H+ pumped from the blood into the ambient water by the Cl– and Na+ pumps are produced by aerobic catabolism, being formed by the reaction of metabolically produced CO2 with H2O (see Figure 28.2). Thus the Na+ and Cl– pumps participate in removal of metabolic wastes.

Because HCO3– and H+ are principal players in acid–

base regulation (see page 664), the Na+ and Cl– pumps

freshwater animals.

ACTivE iON uPTAKE iN FrESHWATEr FiSH: THE gillS AS iON- rEgulATOry OrgANS The gills of teleost fish—although of- ten discussed simply as breathing organs—in fact carry out two major functions that serve homeostasis: In both freshwater and seawater, the gills function both as ion-regulatory organs and as gas-exchange organs. The gills are the principal sites where Na+ and Cl– are taken up by active transport from freshwater, as we have already said. During the early development of freshwater fish, the gills—despite being thought of usually as breathing organs—as- sume their ion-uptake function first. To be more specific, when larvae first hatch out of the egg, both functions are carried out by the general integument, according to studies of rainbow trout

5 Recent research has revealed that an important, deadly fungal pathogen of amphibians, Batrachochytrium dendrobatidis, severely disrupts ion uptake across the skin, and this may be the chief way the fungus kills.

Gill

Water and Salt Physiology of Animals in Their Environments 745

CO2 (from metabolism) +

H2O (from body water) H2CO3

HCO3–

++ H

Bicarbonate ions (HCO3–) and protons (H+) are exchanged for Cl– and Na+, respectively, by the active-transport mech- anisms, which require ATP.

sometimes play critical roles in the acid–base physiology of

FigurE 28.2 ion exchanges mediated by active Na+ and Cl– transport in the gill epithelium of freshwater teleost fish The mechanisms of active transport exist within single epithelial cells.
The view here is a whole-epithelium view and therefore, as discussed in Chapter 5 (see Figure 5.14), does not specify the cell-membrane mechanisms involved.The cell-membrane mechanisms are dis- cussed in Box 5.2.

(Oncorhynchus mykiss) living in freshwater. Responsibility for Na+ uptake from the ambient water shifts to the gills about 15–16 days after hatching, whereas responsibility for O2 uptake doesn’t shift to the gills until 23–28 days.

The two images of a single gill filament in FigurE 28.3 help emphasize the two major functions of the gills in adult teleosts. Figure 28.3A shows the microscopic structure of the filament. It consists of a thin, principal part—shaped somewhat like the blade of a feather—that bears many thin folds, the secondary lamellae, on its upper and lower surfaces. Blood flows through all these parts. The secondary lamellae greatly increase the sur- face area across which O2 can diffuse inward from the ambient water into the blood (see Figure 23.11). Figure 28.3B shows the same filament visualized in a way that reveals the presence and location of the gill epithelial proteins that are instrumental in ion transport between the ambient water and blood; in this specific case (although not always), the epithelial cells expressing the ion-transport proteins are located in parts of the filament other than the secondary lamellae.

The method used to obtain Figure 28.3B—immunocytochem- istry—is worth brief mention before we go further because it is the principal method used at present to study gill ion-transport functions throughout the animal kingdom. Two fluor-labeled antibodies were used: one antibody against Na+–K+-ATPase and another antibody against a cotransporter protein (not an ATPase) termed NKCC-1, which transports Na+, K+, and Cl− ions in fixed ratios during each transport cycle (see page 117). When the filament

Hill Animal Physiology 4E Sinauer Associates
Figure 28.02 12-23-15

Pond water

Blood pla

Cl–
HCO3–

Na+
H+

Gill
epithelial cell

Active Cl– transport

sma
Cl–

Na+

Active Na+ transport

746 Chapter 28

Overall gill structure

Gill arch

Gill filament

Water flow

Secondary lamellae

FigurE 28.3 A single gill filament of an adult teleost fish viewed in two ways that emphasize the two principal gill functions: O2 uptake and ion pumpingThe color drawing shows the overall gill struc- ture.The gill filament seen in (A) and (B) is from a euryhaline species that occurs over wide ranges of salinity in salt marshes, the kil- lifish Fundulus heteroclitus.This specimen was captured in nearly freshwater (salinity 4‰) in coastal Virginia.The images were acquired by confocal microscopy. (A) The structure of the filament. (B) The locations of two ion-transport proteins in a particular plane (optical sec- tion) of the filament: Na+–K+-ATPase (red) and NKCC-1 (green). Cell nuclei are labeled dark blue.Yellow represents places where both transport proteins occurred together (red + green = yellow). See Figure 23.11 for more on gill structure. (A and B courtesy of Aaron M. Florn.)

was exposed to the antibodies, they bound where Na+–K+-ATPase and NKCC-1 occurred, respectively. Then, when the filament was scanned with lasers that excited the fluors, the antibody against Na+–K+-ATPase glowed red, whereas that against NKCC-1 glowed green. Thus red shows where Na+–K+-ATPase was located, green shows where NKCC-1 was located, and yellow (the combination of red and green) shows where both gill epithelial proteins occurred in approximately the same location.

Let’s now focus briefly on the cellular level of organization. The

gill epithelium in fish consists principally of two types of cells: (1)

mitochondria-rich cells (MRCs), also called chloride cells, and (2) pave-

ment cells. The MRCs are considered to be the principal (although

not exclusive) sites of active ion transport in the gills.6 They are a

(B) The ion-transport function of a gill filament: Ion-transport proteins labeled red and green

Figure 28.03 12-23-15

FigurE 28.4 shows a portion of the gill epithelium of a freshwater fish visualized by scanning electron microscopy (the magnification is far greater than in Figure 28.3). In this electron micrograph, we see a single freshwater-type MRC surrounded by pavement cells. Regarding the two major functions of the gills, O2 uptake is believed to occur principally across pavement cells, which usually occupy more than 90% of the gill epithelium and are thinner than MRCs. As for ion uptake, physiologists would like to know precisely where Cl– uptake and Na+ uptake occur, but the problem of deducing the detailed molecular mechanisms of ion transport and localizing them to particular cells is exceedingly challenging, and probably the localization of pumps to cell types will require considerably more research. Current models of Na+ and Cl– uptake in freshwater

Mitochondria-rich (chloride) cell

1 μm

(A) The O2 uptake function of a gill filament:
The expanded surface area for gas exchange

20 μm

fish are discussed in Box 5.2, and modulation of gill function when migratory fish swim between freshwater and seawater is discussed later in this chapter (see page 759).

The number of MRCs in the gill epithelium of a fish living in freshwater is variable and under adaptive (partly hormonal) control. One condition that increases the number of MRCs is alkalosis: excess blood bicarbonate (HCO3–) (see pages 644). During alkalosis,

Pavement cell

central focus of research on ion transport in both freshwater and

Hill Animal Physiology 4E marinefish,andbOx28.1SdiniasuceursAssessotchiaeteirspropertiesanddiversity.

6 Based on the immunocytochemical evidence, the labeled cells in Figure 28.3B are MRCs.

FigurE 28.4 A mitochondria-rich cell surrounded by pave- ment cells in the gill epithelium of a freshwater teleost
fish The image is a scanning electron micrograph of the outer surface of the gill epithelium of a brown bullhead (Ictalurus nebu- losus) that had been living in ordinary freshwater. One pavement cell is outlined (yellow dashed line) to show its limits. (See Box 28.1 for detail on mitochondria-rich cells.) (Photograph courtesy of Greg Goss and Steve Perry; from Goss et al. 1998.)

the MRCs, besides becoming more numerous, also modify their

2

–Figure 28–.05 12-23-15
cell proteins—upregulating a key Cl /HCO3 countertransport

protein that exports HCO3– from the body fluids in exchange for Cl– (see Figure B in Box Extension 5.2).

A second, and fascinating, condition that leads to increased numbers of MRCs in freshwater fish is life in very “soft” water: water of exceptionally low Ca2+ concentration (FigurE 28.5). Freshwater fish acquire most of their Ca2+ from the water in which they live, rather than from their food. The MRCs (or a subset of them) are the sites of active Ca2+ uptake. When fish are living in Ca2+-poor waters, an increase in the number of MRCs is believed to help them acquire sufficient Ca2+. However, increasing the number of MRCs can also interfere with uptake of O2! Recent research on several species has shown that in fish living in very soft water, the replacement of pavement cells by MRCs in the

Hill Animal Physiology 4E Sinauer Associates

FigurE 28.5 Cellular acclimation to living in two types of water in the gill epithelium of freshwater fish Seen here
are sections of the secondary lamellae in the gills of rainbow trout (Oncorhynchus mykiss), viewed using light microscopy and stained to show mitochondria-rich cells. (A) Tissue section from a fish that had been living in ordinary freshwater with a Ca2+ concentration

of 0.4 millimolar (mmol/L). (B) Tissue section from a fish that had been living for 2 weeks in very “soft” freshwater with a Ca2+ concen- tration of 0.05 mmol/L. (Photographs courtesy of Steve Perry; from Perry 1998.)

secondary lamellae can double the average diffusion distance between blood and water in the gills, because MRCs are thick (see Figure 28.5)—thicker than the pavement cells they replace. This doubling of the diffusion distance measurably interferes with O2 uptake. Thus freshwater fish exhibit a trade-off between their ability to take up Ca2+ and their ability to take up O ; increasing one ability

decreases the other.
The concept of trade-offs is a major theme in modern ecology and

evolutionary biology. The situation in freshwater fish just described is one of the physiological trade-offs that, considering all of animal physiology, is best understood at a cellular level.

FOOd ANd driNKiNg WATEr Freshwater animals of all types—fish, crayfish, and so forth—gain ions from their food, in addition to acquiring them by active uptake from the ambient wa- ter. The role of food in meeting ion needs is not well understood, although inputs of ions by active transport are generally thought to exceed those from food. In addition to eating food, freshwater animals also have the opportunity to drink water. But do they? Freshwater animals typically must produce urine at a very high

Water and Salt Physiology of Animals in Their Environments 747 (A) Trout living in ordinary freshwater

(B) Trout living in very “soft” freshwater

Individual secondary lamellae

Mitochondria- rich cell

15 μm

15 μm

Mitochondria- rich cell

748 Chapter 28

rate just to deal with their passive osmotic water influxes. Thus one would not expect them to drink, and usually they do not. However, recent studies of teleost fish in freshwater have revealed that some species—especially when they are larvae—do drink sufficiently to raise their total water influx by 5%–50% more than their osmotic influx alone. The reasons for this drinking remain obscure.

quANTiTATivE ExAMPlE ANd COST ESTiMATES The usual pattern of water–salt balance in freshwater animals is summarized in Figure 28.1B (showing a crayfish) and in Figure 28.8A (showing a fish). To review this pattern in words, let’s look quantitatively at the gains and losses of water and Na+ in a freshwater crayfish (Astacus). When fasting at 20oC, a 29-g crayfish excretes about 2.4 mL of urine per day—indicating that it gains 2.4 mL of water per day by osmosis, principally across its gills. The crayfish’s urine is very dilute in Na+ (1 mM Na+). Therefore only about 2–3 micro- moles (μmole) of Na+ is lost per day in its urine. The animal’s loss of Na+ by direct diffusion into the surrounding water is much greater, approximately 240 μmole/day. The Na+ lost by excretion and diffusion is replaced by active Na+ uptake across the gills at a rate near 240 μmole/day.

According to a relatively recent study using modern methods, the energy cost of osmotic–ionic regulation in freshwater animals is about 3%–7% of their resting metabolic rate. The study—which focused on rainbow trout and mudpuppies (aquatic amphib- ians)—was based on measurements of ion-pumping rates in the gills, skin, and kidneys, plus information on the ATP demands of the ion pumps.

A few types of freshwater animals exhibit exceptional patterns of regulation

The typical pattern of osmotic–ionic regulation we’ve described is not observed in all freshwater animals. As usual, a look at excep- tions can be as conceptually revealing as a look at the rule. Here we will look just at those exceptional freshwater animals that fail to produce a dilute urine.

The freshwater crabs are outstanding examples. These animals are unfamiliar to most North American readers because historically they have not occurred in North American waters.7 Nonetheless, freshwater crabs are common on most other continents, usually at tropical or subtropical latitudes. Two species that have been studied are Potamon niloticus, an African crab, and Eriocheir sinensis, found in Asia and Europe. Freshwater crabs typically maintain high blood osmotic pressures in comparison with other freshwater animals: about 500–650 mOsm in Potamon and Eriocheir, for ex- ample (compare Table 28.1). As usual, the major blood solutes are Na+ and Cl–. The feature that makes freshwater crabs distinctive is that their urine is virtually isosmotic to their blood plasma. Its ionic composition is also very similar to that of blood plasma. Consequently the loss of Na+ and Cl– per unit of volume of urine in the freshwater crabs is very high by comparison with that in most freshwater animals. How do the crabs compensate? One well-known part of the answer is that the bodies of freshwater crabs exhibit extraordinarily low permeability to water. Because

7 The freshwater crab Eriocheir has recently been introduced into some river systems along the West Coast of the United States and is now established there as an alien species.

of this low permeability, the crabs experience relatively low rates of osmotic water influx, and therefore the rates at which they must excrete their high-concentration urine are low. Potamon and Eriocheir, for instance, excrete water equivalent to only about 0.6%–3.6% of their body weight per day (compare Table 28.2). The unusually low urine output of the freshwater crabs—which results from their unusually low permeability to water—helps limit the rate at which they lose ions. The total quantities of Na+ and Cl– that the crabs lose—by both diffusion and urine excretion—although large by comparison with the quantities lost by freshwater crayfish, are “manageable,” in the sense that the ions can be replaced by active uptake (across the gills) from the ambient water.

A few exceptional teleost fish living in freshwater also excrete urine that is nearly isosmotic to their blood plasma. One well-studied example is the toadfish Opsanus tau, a primarily marine fish that enters freshwater creeks. The freshwater crabs and the toadfish seem likely to be relatively recent immigrants to freshwater. Their short evolutionary history in freshwater helps explain why their kidneys have not evolved the ability to make dilute urine.

Why do most freshwater animals make dilute urine?

The freshwater crabs and the toadfish prove that production of dilute urine is not a necessity for life in freshwater; the produc- tion of relatively concentrated urine increases ion losses per unit of volume, but if total urinary losses can be restrained enough that ions can be replaced, existence in freshwater is possible. Why, then, have the vast majority of freshwater animals evolved the capacity to make urine that is dramatically hyposmotic to their blood plasma?

The answer is probably energy savings. To produce a dilute urine, the kidneys start with a fluid that is as concentrated as blood plasma and actively extract NaCl from it. Every Na+ or Cl– ion removed from the urine prior to excretion is an ion that does not have to be replaced by active uptake from the ambient water. In the urine—as it is formed in the kidneys—the concentrations of Na+ and Cl– are initially as high as in the blood plasma; only gradually—as the ions are reabsorbed—do urine ion concentrations fall to low levels. In contrast, the concentrations of Na+ and Cl– in the ambient water are always very low. Two lines of argument—one based on thermodynamic principles and the other on the molecular details of transport mechanisms—indicate that active uptake of Na+ or Cl– from a relatively concentrated source costs less energy per ion than uptake from a dilute source. Thus removing ions from urine prior to excretion is less costly than replacing the same ions from the ambient water.

Animals in the Ocean

Animal life in the oceans is far more phylogenetically diverse than that in the other major habitats on Earth, probably in good part because animals originated in the oceans. Today, all phyla and most classes of animals have marine representatives.

Many phylogenetic groups of animals moved from the oceans to freshwater and the land over the course of evolutionary time. In turn, many freshwater and terrestrial groups reinvaded the oceans. Consequently, whereas some modern marine animals have a continuously marine ancestry, others trace their history to forms that occupied other major habitats.

Such a phylogenetic history has sometimes left major imprints on the water–salt physiology of modern marine animals. For example, although the octopus and the marine teleost fish seen swimming in seawater in FigurE 28.6 may look like they would have similar blood osmotic concentrations, their concentrations actually are very different, as we will see in the following sections. History is believed to be the explanation. Whereas octopuses probably trace a continuously marine ancestry, marine teleost fish are likely descended from freshwater ancestors.

(A)

Water and Salt Physiology of Animals in Their Environments 749 Most marine invertebrates are

isosmotic to seawater

Most marine invertebrates are isosmotic, or nearly so, to seawater. Included are the marine molluscs—exemplified by the octopus in Figure 28.6A—and such other marine animals as sponges, coelen- terates, annelids, echinoderms, and most arthropods. For the most part, these animals are products of lines of evolution that never left the sea. They have always lived in seawater, and this probably explains why they have the simplest possible osmotic relation to seawater. The osmotic pressure of seawater is about 1000 mOsm, and the osmotic pressure of marine invertebrates’ body fluids is about the same. Being essentially isosmotic to their environment, marine invertebrates do not tend to gain or lose water by osmosis to any great extent: They do not face problems of osmotic regulation.

The solutes in the blood plasma of marine invertebrates are mostly inorganic ions, and the ionic composition of their blood plasma tends to be grossly similar to that of seawater. Despite this similarity, as exemplified in TAblE 28.3, the ionic composition of the blood plasma seems universally to differ in detail from the ionic composition of seawater. A particular ion often proves to be relatively concentrated in some animal species but relatively dilute in others; for example, Mg2+ is relatively high in concentration in the blood plasma of the squid Loligo but low in that of the crab Carcinus. The adaptive significance of such differences in blood ionic composition is generally unknown.

Isosmotic marine invertebrates maintain the differences in ionic composition between their blood plasma and seawater by ionic regulatory processes. These animals are typically relatively permeable to ions (and water). Ions therefore tend to diffuse between their blood and seawater with ease, following their electrochemical gradients. One process these animals commonly use to maintain their blood ionic composition is active uptake of ions from seawater at the body surface or from ingested seawater

(B)

FigurE 28.6 Two ocean animals with different blood osmotic pressures, an invertebrate with blood isosmotic to seawater and a teleost fish with blood dramatically hyposmotic to seawater (A) The octopus (Octopus cyanea) belongs to the group of marine molluscs known as cephalopods, which also includes squids and cuttlefish. (B) The teleost fish at the right is Zebrasoma ve- liferum. Both species shown here are found in Hawaii.

750 Chapter 28

Source: After Potts and Parry 1964.

in the gut. A second common process is kidney regulation of blood composition. In crustaceans, molluscs, and some other groups, although the excretory organs make a urine that is approximately isosmotic to the blood plasma, they alter the urine’s ionic com- position, thereby contributing to ionic regulation. For example, in most marine decapod crustaceans, the urine is richer in Mg2+ and SO42– than the blood plasma (ionic U/P = 1.1–4.2 in several species), which helps keep plasma concentrations of these ions lower than seawater concentrations (see Figure 27.7).

Hagfish are the only vertebrates with
blood inorganic ion concentrations that make them isosmotic to seawater

The hagfish, an exclusively marine group of jawless primitive vertebrates, resemble the great majority of marine invertebrates in two key respects: (1) Their blood is approximately isosmotic to seawater, and (2) their blood solutes are principally Na+, Cl–, and other inorganic ions (see Table 28.3). The ionic regulatory processes of hagfish are similar to those of osmoconforming marine inverte- brates. Hagfish appear to be the only modern vertebrates that trace a continuously marine ancestry (see Box 28.2).

The marine teleost fish are markedly hyposmotic to seawater

As stressed at the start of this chapter, the marine teleost (bony) fish are hyposmotic regulators: Their blood osmotic pressures are far lower than the osmotic pressure of the seawater in which they swim. As in freshwater teleosts, Na+, Cl–, and other inorganic ions constitute most of the solutes in the blood plasma. One of the most intriguing questions about marine teleosts concerns the origin

of their hyposmotic state: Why is their blood plasma dramatically more dilute in ions and lower in osmotic pressure than seawater? Most biologists conclude, as discussed in bOx 28.2, that the dilute body fluids of marine teleosts are an evolutionary vestige: These fish are generally believed to be descended from ancient ancestors that lived in freshwater.

The blood osmotic pressures of marine teleost fish are typically 300–500 mOsm—higher than those of freshwater teleosts (about 250–350 mOsm), but not exceptionally so. Evidently, when the teleost fish invaded the oceans from freshwater in the course of their evolution, they evolved modest increases in the concentration of total blood solutes. This change served to reduce the difference in concentration between their blood plasma and the environmental water (the seawater) in their new habitat.

Despite such a change, today’s marine teleost fish—because of their profoundly dilute state relative to seawater—face a difference between their blood plasma and their environmental water that is far greater than that faced by freshwater teleosts. In freshwater teleosts, blood osmotic pressure averages about 300 mOsm higher than the osmotic pressure of freshwater. In marine teleosts, however, the difference between the blood and environmental osmotic pressures is about 600 mOsm: approximately twice as great!8 This

consideration in itself would tend to saddle the marine fish with a relatively high rate of osmotic water flux. Marine teleosts, however, are typically less permeable to water than freshwater teleosts, so in fact the osmotic fluxes experienced by the two groups are roughly similar in magnitude (for a given body size).

Of course, although the osmotic fluxes of marine and freshwater teleosts may be similar in magnitude, they are totally opposite in direction. Because a marine teleost is hyposmotic to seawater, water tends to leave its body osmotically, rather than entering as it does in a freshwater teleost. For a hyposmotic animal, the ocean is a desiccating environment. + – 2+ 2–

The concentrations of Na , Cl , Mg , SO4
inorganic ions are far lower in the blood plasma of marine teleosts than in seawater, suggesting that marine teleost fish also face problems of inward diffusion of multiple ions. Moreover, the concentration gradients between the blood plasma and the envi- ronment for the two major plasma ions, Na+ and Cl–, are large by comparison with the (oppositely directed) gradients seen in fresh- water teleosts. Nonetheless, actual rates of ion diffusion depend on electrical gradients and gill permeability, not just on ion concentration gradients. When all these factors are taken into account, inward Na+ diffusion turns out not to be much of a problem for marine teleosts (or may not even occur in some species), because the gill epithelium is positively charged on the inside, repelling Na+. In contrast, some other ions—most notably Cl–—tend to diffuse into the blood plasma of marine teleosts from seawater at substantial rates, tending to concentrate the body fluids of the fish.

8 Average blood osmotic pressure in marine teleosts is about 400 mOsm, and the osmotic pressure of seawater is about 1000 mOsm—a difference of 600 mOsm.

, and some other

Water and Salt Physiology of Animals in Their Environments 751

rEPlACEMENT OF WATEr lOSSES Marine teleost fish lose water by osmosis and, to a lesser extent, by urine production. To replace the water they lose (and thereby volume regulate), these fish drink seawater. Although some drink an amount of seawater that is less than 1% of their body weight per day, others drink more than 50% per day, and the average is probably 10%–20% per day.

At first sight, drinking seawater seems to be a straightforward way to obtain water. Consider, however, that when seawater is first taken into the gut of a marine teleost, it is strongly hyperosmotic to the fish’s blood plasma. Consequently, H2O is predicted to travel by osmosis out of the blood plasma into the ingested seawater in the gut, not vice versa—and that is exactly what happens. Studies of several species show that as ingested seawater travels through the esophagus, stomach, and (in at least some instances) anterior intestine, not only do Na+ and Cl– diffuse into the blood across the gut wall, but also H2O enters the gut fluids by osmosis. Gradually, therefore, the ingested seawater in the gut expands in volume and is diluted. Water uptake from the gut fluids eventually occurs, nonetheless. This is true because in later parts of the intestine, Na+ and Cl– are actively transported out of the gut contents into the blood. This ATP-requiring, active uptake of Na+ and Cl– into the blood creates conditions that favor the osmotic uptake of water. In the simplest cases to understand, the active uptake of the ions from the gut fluids renders the gut fluids hyposmotic to the blood. Often, however, a process called near-isosmotic fluid transport occurs, in which the gut fluids and blood plasma remain approximately isosmotic as water moves briskly by osmosis into the blood; in

this case, highly localized osmotic gradients within the epithelium of the intestines are involved in translating ion uptake into water uptake (termed local osmosis). Recent evidence, discussed more on page 760, indicates that aquaporins in the intestinal epithelium are instrumental in facilitating water uptake from the gut in fish in seawater.

By the time ingested seawater is completely processed, about 50%–85% of the H2O in the seawater is absorbed into the blood. However, a much greater proportion of the NaCl in the ingested seawater—often more than 97%—is absorbed. This is true because NaCl absorption is required to drive the absorption of H2O. The influx of NaCl into the blood aggravates the problems of Na+ and Cl– regulation that the fish face. Accordingly, in marine teleosts—as in freshwater teleosts—the process of volume regulation worsens the problems of ionic regulation.9

Divalent ions10 in ingested seawater are handled very differ- ently from the monovalent ions, Na+ and Cl–. The gut epithelium is poorly permeable to the major divalent ions, Mg2+ and SO42–. Consequently, although the divalent ions diffuse into the blood to a small extent as seawater passes through the gut, for the most part they remain in the gut and are expelled in the feces.

9 Volume regulation and ionic regulation are inextricably linked in the marine and freshwater teleosts because the major solutes of the body fluids in both of these groups of fish are inorganic ions.

10 Divalent ions have two charges per ion, either two positive charges or two negative charges.

752

Chapter 28

Pavement cells

Golgi apparatus

Mitochondria

Nucleus

Intracellular tubular system

Basement membrane 2.5 μm

Apical crypt

Mitochondria-rich cells are dense with mitochondria— accounting for their name.

Mitochondria-rich cells have an extensive intracellular tubular system, composed of branching tubules, continuous with the basal and lateral portions
of the cell membrane. Although the parts of the tubular system seen in this section through the

cell appear to be disconnected, the parts are believed to be mostly interconnected in intact cells.

Non-differentiated cells

FigurE 28.7 A section of a mitochondria-rich cell (chloride cell) of a marine teleost fish In some species of marine teleosts, MRCs occur not just in the gill epithelium but also in the inner opercular epithelium, jaw epithelium, and certain other surfaces, implicating these other body parts— as well as the gills—in ion excretion.The MRC shown here is from the opercu- lar epithelium of a seawater-acclimated killifish, Fundulus heteroclitus. (After Degnan et al. 1977.)

uriNE We now turn to the question of how marine teleosts elimi- nate the excess ions that enter their body fluids from the gut, or that diffuse into their body fluids from seawater across their gills or other external body surfaces. For the most part, excess divalent ions in the body fluids are removed by excretion in the urine, whereas excess monovalent ions are excreted by the gills.

The kidneys of marine teleosts typically excrete urine that is about as concentrated as they can possibly produce, approximately isosmotic to the blood plasma.11 The fact that the osmotic pressure of the urine matches that of the plasma (osmotic U/P ≅ 1) means that the excretion of urine cannot help the fish with their osmotic regulatory problem (see Figure 27.7). However, the ionic composition of the urine differs dramatically from that of the plasma, and the kidneys are the principal organs that carry out ionic regulation of Mg2+, SO42–, and Ca2+. Whereas U/P ratios for Na+, Cl–, and K+ are below 1, those for Mg2+ and SO42– are far greater than 1. The kidneys thereby void the major divalent ions preferentially in relation to water and keep

Hill Animal Physiology 4E
plasma concentrations of those ions from increasing.

Sinauer Associates

into the surrounding ocean. The excretion of Cl– is active and is carried out by seawater-type mitochondria-rich cells (MRCs) in the gill epithelium (FigurE 28.7). These cells are often called chloride cells in the study of marine teleosts because of their well-established excretion of Cl–. In fish soon after hatching, as discussed in Chapter 4 (see Figure 4.6), the MRCs are principally found in the general integument, but soon the cells become local- ized to the gills. bOx 28.3 outlines the mechanism these cells employ to pump Cl–. Although the gill epithelium is believed always to transport Na+ as well as Cl– out of the blood into the seawater, the excretion of Na+ occurs by mixed mechanisms; Na+ excretion is probably active in about half the species that have been studied, but passive in the others (in which Na+ diffuses outward, attracted by an outside-negative electrical gradient generated by active Cl– excretion). The elimination of Cl– and Na+ by the gills of marine teleost fish provides our first example of extrarenal salt excretion: excretion of inorganic ions by struc- tures other than the kidneys.

Present evidence indicates that excretion of NaCl by the gills in many teleosts is accomplished without concomitant excretion of water; the material excreted is purely ions. Thus, in addition to voiding NaCl from the blood (ionic regulation), the process produces a fluid that is essentially infinitely higher in osmotic pressure than the blood plasma. The process therefore tends to lower the osmotic pressure of the plasma (see Figure 27.7) and maintains the blood osmotic pressure at a level below the ambient osmotic pressure. The gills are in fact the sites where osmotic regulation is principally accomplished.

quANTiTATivE ExAMPlE ANd COST ESTiMATES In FigurE 28.8, the pattern of water–salt regulation in marine teleost fish is summarized (see Figure 28.8B) and contrasted with the pattern in freshwater teleost fish (see Figure 28.8A). Let’s review the pattern in marine teleost fish by making use of quantitative data for one particular species that has been thoroughly studied, the southern flounder (Paralichthys lethostigma). An individual flounder that weighs about 1 kg loses water equivalent to about 7.9% of its body

Figure 28.07 12-23-15

For every milliliter of water that is first ingested and absorbed

and then excreted as urine, a marine teleost is left with an excess of solutes because, although the water enters its body hyperosmotic to its body fluids, the water leaves its body isosmotic to its body fluids. From the viewpoint of osmoregulation, therefore, production of urine by marine teleost fish is an outright liability, and we would expect the fish to limit their volume of urine to the minimum necessary for excretion of solutes that are not excreted by other routes. Nitrogenous wastes and the principal ions, Na+ and Cl–, are voided across the gills. Thus the role of the kidneys in marine teleosts is largely limited to excretion of divalent ions, and the rate of urine production can be low. The urine volumes of several species have been measured to be just 0.5%–3.5% of body weight per day (compare Table 28.2).

ExTrArENAl NaCl ExCrETiON by THE gillS The gills of an adult marine teleost assume primary responsibility for excreting excesses of the major ions, Na+ and Cl–, from the blood plasma

11 The kidneys of fish are incapable of producing urine that is hyperosmotic to blood plasma.

Basolateral cell membrane

(A) Freshwater teleost

Salt loss by diffusion

Salts and water in feces

Large amounts of urine, very hyposmotic to plasma

Water and Salt Physiology of Animals in Their Environments 753 (B) Marine teleost

Water uptake by osmosis

Salt gain by diffusion

Water loss by osmosis

Gills

Salts and water in food (generally do not drink)

Gills

Salts and water in food

Salts and water in seawater ingested (source of net water gain)

Hyperosmotic to ambient water

FigurE 28.8 Contrasting water–salt relations in freshwater and marine teleost fish

weight per day because of osmosis from its body fluids into the surrounding seawater. To replace this water, and also to replace urinary water losses of 0.4% of body weight per day and fecal losses of 2.7% per day, the fish drinks seawater equivalent to 11% of its body weight per day. From the seawater it ingests, the fish absorbs 76% of the H2O, but in doing so it absorbs much higher percentages of the Na+ (99%) and Cl– (96%). The flounder produces a scanty urine having a U/P ratio for Mg2+ of about 100 and a U/P ratio for SO42– of 330; the urine removes excesses of both of these divalent ions from the body fluids. The gills of the flounder excrete virtually all of the excess monovalent ions, Na+ and Cl–.

The energy cost of Na+ regulation, Cl– regulation, and osmotic regulation in marine teleosts has been estimated in several species (including tunas) to be 8%–17% of the resting metabolic rates of the fish, based on measured ion-pumping rates and the known ATP costs of pumping. Rainbow trout—which can live in freshwater or seawater—are estimated to devote 8% of their resting energy use to Na+, Cl–, and osmotic regulation when living in seawater, but 3% when living in freshwater. The higher cost in seawater reflects the fact, earlier stressed, that for a teleost fish, the difference in concentration between the blood and the ambient water is about twice as great in seawater as in freshwater.

Active uptake of Na+ and Cl–

Salts and water in feces

Small amounts of urine, nearly isosmotic to plasma, rich in Mg2+ and SO42–

Active extrusion of Cl–, active or passive outflux of Na+

Hyposmotic to ambient water

754 Chapter 28

Some arthropods of saline waters are hyposmotic regulators

Quite a few arthropods that live in the ocean or more-saline waters, such as salt lakes, maintain their blood osmotic pressure at a level hyposmotic to the water in their environment. These animals in- clude brine shrimp (“sea monkeys”), the insects that live (usually as larvae) in salty waters, and some marine crabs and shrimps. Biolo- gists generally believe that the ancestors of most of these animals lived in more-dilute habitats, and their body fluids bear an imprint of that earlier time. Their mechanisms of hyposmotic regulation have been well studied in a few cases and usually parallel those of marine teleost fish.

limiting water losses in the dehydrating terrestrial environment, so they tend to exhibit low integumentary permeabilities.

These animals nonetheless confront problems of water loss and salt loading. They lose water, for example, by pulmonary evaporation during breathing; they also lose water to some extent across their skin, not only when they are immersed in seawater, but also when they are exposed to the air. These animals often gain excess salts from the foods they eat; for example, when they prey on marine plants or invertebrates that are isosmotic to seawater, they ingest body fluids that have far higher salt concentrations than their own. In addition, they probably often take in quantities of seawater with the foods they eat, although, for the most part, they are thought not to drink seawater.

Hill Animal Physiology 4E Sinauer Associates

Marine reptiles (including birds) and mammals

Box Figure 28.03 12-23-15

are also hyposmotic regulators

The marine turtles and lizards—in common with other nonavian reptiles—are generally not able to produce urine that is more concentrated in total solutes than their blood plasma. The urine-concentrating capabilities of marine birds are incompletely understood, but for most species the maximum urine concentration appears to be isosmotic to the blood plasma or only modestly hyperosmotic (U/P ≤ 2). Because of these meager concentrating abilities, the kidneys of marine turtles, lizards, and birds are in general not able—by themselves—to maintain the

blood of the animals hyposmotic to seawater (see Figure 27.7). Obviously, then, these animals require a way to excrete salts in a more concentrated state than their kidneys can provide. Virtually

The sea turtles, sea snakes, penguins, gulls, whales, seals, and other marine reptiles and mammals—like marine teleost fish—are markedly hyposmotic to seawater. All are descended from terrestrial ancestors, and their blood compositions are clearly carryovers from their ancestors. The blood osmotic pressures of all these marine vertebrates tend to be about 400 mOsm: just modestly higher than the values seen in modern-day terrestrial and freshwater vertebrates.

Because the marine nonavian reptiles, birds, and mammals are air breathers, they do not expose permeable respiratory membranes to seawater. Another advantage of their terrestrial heritage is that they have inherited integuments that were originally adapted to

MAriNE rEPTilES (iNCludiNg birdS)

(A) Herring gull

Water and Salt Physiology of Animals in Their Environments 755

Each gland consists of many longitudinal lobes, each of which contains a great many branching, radially arranged secretory tubules that discharge into a central canal.

Salt gland

Salt-gland secretions of birds (and lizards) exit by way of the nostrils, which are positioned high on the bill in tube-nosed birds such as fulmars, but at the end of the bill in most birds.

FigurE 28.9 Avian salt glands
cated above the eyes. Ducts carry the secretions of the salt glands to the nasal passages, and the secretions drip out from the external nares (nostrils). (A) The structure of the salt glands of a herring gull. Each salt gland lies in a shallow depression in the skull above the eye. (B) A northern fulmar—a type of oceanic bird—showing the out- lines of the salt glands above the eyes and the dripping of salt-gland secretions from the nostrils. (A after Schmidt-Nielsen 1960; B after Goldstein 2002.)

all of them have salt glands, organs of extrarenal salt excretion that make up for the meager concentrating abilities of their kidneys.12 These glands are located in the head, as illustrated for birds

in FigurE 28.9. They put out secretions that are dramatically hyperosmotic to the blood (by a factor of four to five in many spe- cies). Moreover, these secretions (as shown in TAblE 28.4) contain concentrations of Na+ and Cl– (and K+ as well) that exceed those in seawater. Thus birds, lizards, and turtles with salt glands are, in principle, able to extract pure H2O from seawater; they could drink seawater and void the major monovalent ions in less H2O than they ingested, retaining the excess H2O in their bodies. The salt gland secretions are discharged into the nasal passages in birds (see Figure 28.9B) and lizards. In sea turtles the secretions are emitted like tears. The cellular mechanism of salt secretion by

12 Salt glands have been reported in marine lizards (e.g., the Galápagos iguana), sea turtles, marine snakes, and 14 orders of marine birds. However, they have not been reported in the passerine (“perching”) birds.

Lobe

Central canal

(B) Northern fulmar

4E

The salt glands of birds are lo-

Sources: Schmidt-Nielsen and Fange 1958; Schmidt-Nielsen 1960; Marshall and Cooper 1988.

the salt glands conforms to the model in Box 28.3, at least in birds and sea turtles.

The ingestion of a salt load by an animal with salt glands is promptly followed by an increase in the rate of secretion by the glands. Control of this response, at least in birds, is mediated prin- cipally by the parasympathetic division of the autonomic nervous system. When osmoreceptors located in or near the heart and brain detect high blood osmotic pressures, the parasympathetic nervous system releases acetylcholine in the salt glands; this chemical mes- sage induces gated Cl– and K+ channels in the mitochondria-rich cells (chloride cells) of the salt glands (see figure in Box 28.3) to open, activating secretion.

In addition to these acute responses, salt glands also undergo chronic responses (acclimatization). For example, if an individual bird experiences a chronic increase in salt ingestion—as it would after migrating from a freshwater habitat to an ocean habitat—its salt glands typically increase in size, concentrating ability, and peak secretory rate. These changes reverse if the bird returns to freshwater.

The tears observed flowing down the faces of sea turtles when they emerge onto beaches to lay eggs are of some renown. We now understand that they are secretions of salt glands, not tears of emo- tion. If you watch a fulmar or gull standing by the ocean, you will see—emerging from its nostrils—droplets of salt-gland secretions (see Figure 28.9B), often flicked away by a shake of its head.

Sea snakes, it has recently been discovered, differ in their water–salt relations from other marine reptiles, even though they have salt glands (which empty into the mouth). Based on study of

756 Chapter 28

Sources: Hill and Wyse 1989; Pillans et al. 2005.

one common species (Hydrophis platurus), the salt glands are not effective enough to prevent dehydration. The snakes spend long periods in a relatively dehydrated state, and to rehydrate, they need to drink freshwater (or other dilute water).

MAriNE MAMMAlS Mammals, as a group, are capable of pro- ducing the most concentrated urine of all vertebrates. This ability is believed to be the key for marine mammals such as seals and whales to display hyposmotic regulation. Salt glands or other mechanisms of extrarenal salt excretion are not known in mammals.

As important as the kidneys are in marine mammals, the urine-concentrating abilities of these animals are not dramatic; their concentrating abilities are not particularly high in comparison with those of nonmarine mammals of similar body size. Partly for this reason, the overall patterns of water and salt balance in seals and whales remain open to debate. Existence on a diet of teleost fish poses no great challenges. To date, however, the data available leave unclear whether or how most species could exist while chronically eating only invertebrates, which often have substantially saltier body fluids than fish.

Another area of uncertainty is drinking. Although seals and whales are thought generally not to drink seawater, research in the last 25 years has revealed that some species of both groups do drink under certain circumstances; some fur seals, for instance, drink sea- water when hauled out on land for weeks in hot climates during their breeding season. Physiologists are debating the potential advantages of drinking for animals that in general cannot concentrate salts in

their urine to levels higher than the concentrations seen in seawater (see page 733).

Marine elasmobranch fish
are hyperosmotic but hypoionic to seawater

The marine sharks, skates, and rays—collectively known as the elasmobranch fish—have evolved a novel solution to the osmotic problems of living in the sea. Their blood concentrations of inorganic ions are similar to those of marine teleost fish and well below those in seawater. However, the osmotic pressure of their blood is slightly higher than that of seawater. As illustrated by the sharks in TAblE 28.5, these fish are able to be hyperosmotic to seawater—even though their blood has far lower concentrations of inorganic ions than seawater— because their blood plasma and other body fluids have high concentrations of two organic solutes: urea and, to a lesser extent, trimethylamine oxide (TMAO).13 Because their blood is hyperosmotic to seawater, the marine elasmobranchs experience a small osmotic influx of water, in sharp contrast to the marine teleosts, which confront relent- less osmotic desiccation. The hyperosmoticity of the elasmobranchs—caused by their high blood concentrations of urea and TMAO—is, in effect, a mechanism for obtaining water.

Elasmobranchs are specialized to produce and retain urea. In sharp contrast to teleosts, elasmo- branchs typically synthesize urea as their principal nitrogenous product of protein catabolism (bOx 28.4). Of all the thousands of species of teleosts, fewer than ten are known to employ urea in this role; the others employ ammonia. In contrast, the use of urea as the principal nitrogenous product is universal in marine elasmobranchs. Urea accumulates in the body fluids of marine elasmobranchs because of specializations of their kidneys and gills. Elasmobranchs reabsorb urea from their urine as the urine forms in their kidneys, possibly by use of active urea transport. Moreover, the gills of marine elasmobranchs also retain urea because they have a dramatically low permeability to urea and, according to recent discoveries, they actively return outgoing urea to the blood plasma. Urea in high concentrations can alter the structures of proteins, and the concentration of urea is kept low in most vertebrates (about 2–7 mM in human plasma). Plasma concentrations of urea in marine elasmobranchs—usually 300–400 mM—are “out of sight” by comparison. Some enzymes and other macromolecules in elasmobranchs have evolved exceptional resistance to urea’s denaturing effects. Some elasmobranch organs, such as the heart,

have in fact become dependent on urea for proper function. Recent studies have revealed, however, that many elasmo- branch proteins are just as sensitive to urea’s denaturing effects as homologous proteins in other vertebrates. How can this be? A key part of the answer is that TMAO serves as a counteracting solute. In the amounts present, TMAO offsets the effects of urea (see Figure

13 For the chemical structures of urea and TMAO, see Figure 29.24.

27.10), evidently by opposing effects of urea on deleterious interac- tions of proteins with solvent water, interactions that if unopposed cause protein unfolding.

In most aquatic animals, the blood osmotic pressure is attributable primarily to inorganic ions dissolved in the blood plasma. Because of this, problems of osmotic and ionic regulation are related in particular ways: If an animal tends to gain water by osmosis, it tends to lose ions by diffusion, and vice versa.

about its possible advantages over the “teleost strategy.”14 Until recently, the usual conclusion was that the elasmobranch strategy costs less energy because marine elasmobranchs are able to obtain H2O by “cost-free” osmosis, whereas marine teleosts must drink seawater and pump NaCl out of it to get H2O. The error in this view, we now recognize, is that the osmosis of water into a marine elasmobranch is not “cost-free.” To keep its blood hyperosmotic

Water and Salt Physiology of Animals in Their Environments 757

14 The assumption is that marine elasmobranchs and teleosts both
Hill Animal Physiology 4E inherited low blood salt concentrations from freshwater ancestors, but they

These relations are uncoupled in the marine elasmobranch
fish because about 40% of the blood osmotic pressure is attribut- have diverged in how they manage the consequences.

Sinauer Associates
able to urea and TMAO rather than inorganic iBoonxsF. iBguercea2u8s.0e4th1e2-23-15

elasmobranchs are slightly hyperosmotic to seawater, they tend to gain water by osmosis, but because their blood ion concentrations are below those in seawater, they also tend to gain excess ions by diffusion from seawater. As a consequence of the fact that water enters elasmobranchs osmotically, they need not drink to obtain water, and therefore—unlike teleosts—do not incur the NaCl load caused by drinking seawater (see page 751).

Excess salts are removed from the body fluids of elasmobranchs by the kidneys and, extrarenally, by rectal salt glands. The salt glands, consisting of thousands of secretory tubules, void into the rectum a secretion (see the data for the dogfish shark in Table 28.5) that is isosmotic to the blood, but contains only traces of urea and approximates or exceeds seawater in its concentrations of Na+ and Cl–. The mechanism of NaCl secretion is as described in Box 28.3. Whether active ion excretion occurs across the gills is an unresolved question.

Ever since the “elasmobranch strategy” of water–salt regulation in the sea (FigurE 28.10) was discovered, biologists have speculated

Salt gain by diffusion across gills

Water gain by osmosis across gills

Salts and water in food (generally do not drink)

Rectal-gland secretions rich in NaCl, plus salts and water in feces

Modest amounts of urine, modestly hyposmotic to plasma, rich in Mg2+ and SO42–

The roles of the gills in salt excretion are uncertain

Hyperosmotic but hypoionic to ambient water

FigurE 28.10 Water–salt relations in a marine shark Protein-rich foods are required for adequate urea synthesis.

758 Chapter 28

to seawater, an elasmobranch must synthesize urea, which costs more ATP (see Box 28.4) than merely making ammonia from waste nitrogen (as most teleost fish do). The elasmobranch might also need to pay ATP costs to recover urea from its urine and intercept urea diffusing outward across its gills. In a careful analysis, Leonard Kirschner concluded that the costs of the elasmobranch and teleost strategies are essentially the same. At least in terms of energy, the strategies seem to be “different but equal.”

About 15% of the elasmobranch species alive today occur in dilute brackish waters or in freshwater. Although some are permanent residents of freshwater, most also occur in the ocean. A well-studied example of the latter is the bull shark (Carcharhinus leucas), famed for its rare but devastating attacks on coastal swimmers. When the elasmobranchs that live in the ocean venture into dilute waters, they lower their blood urea concentrations somewhat by decreasing urea synthesis and retention. Nonetheless, they retain elevated blood urea concentrations, as illustrated by bull sharks (see Table 28.5), even though doing so promotes osmotic uptake of excess water in dilute environments.

In addition to marine elasmobranchs, two other types of marine fish maintain high blood concentrations of urea and TMAO: the coelacanth and the holocephalans (chimaeras). The coelacanth (see Table 28.5) is a particularly interesting case because it is the only living example of the crossopterygian fish, the presumed ancestors of the terrestrial vertebrates.

Animals That Face Changes in Salinity

Many aquatic animals face large changes in the salinity of the wa- ters they occupy during their lifetimes. These include (1) animals such as salmon and eels that migrate long distances between riv- ers and the open ocean and (2) animals that live near the margins of the continents. Along coastlines, waters of intermediate salin- ity—brackish waters—occur in estuaries, salt marshes, and other settings (see Chapter 27). Ocean animals that venture into brackish coastal waters encounter lower salinities than they experience when living in the open ocean. Freshwater animals face elevated salini- ties when they enter brackish waters. Some species live principally within estuaries; they face changes in salinity as they move from place to place (see Figure 27.4) or as tides or other water movements shift the waters around them.

In their relations to changing salinities, animals are often cat- egorized as stenohaline or euryhaline. Stenohaline species are able to survive within only narrow ranges of ambient salinity. Euryhaline species, in contrast, can survive within broad ranges of salinity.

Animals are also classified as osmoconformers or osmoregulators (see Figure 27.3). Osmoconformers—sometimes described as poikilosmotic—permit their blood osmotic pressure to match the ambient osmotic pressure. Osmoregulators—sometimes called homeosmotic—maintain a relatively constant blood osmotic pressure even as the ambient osmotic pressure rises and falls.

Most species of invertebrates that occur in the open ocean are stenohaline osmoconformers; when they are placed in brackish waters, their blood osmotic pressure falls, and because they cannot tolerate blood osmotic pressures much lower than those they have in seawater, they do not prosper or may die. Certain marine osmo- conformers are exceptional, however, in that they are euryhaline. Oysters and mussels provide outstanding examples; despite being osmoconformers, some species thrive over wide ranges of salin- ity, from seawater itself to waters less than 20% as concentrated as seawater. For osmoconformers to be so euryhaline, their cells must have remarkable abilities to function over wide ranges of blood osmotic pressure. The cells of euryhaline osmoconformers are noted for having dramatic powers of cell-volume regulation (see Figure 27.8).

An intriguing and commercially important illustration of how water–salt physiology can feature in the lives of euryhaline osmoconformers is provided by the story of MSX, a debilitating protistan parasite of the commercial oyster (Crassostrea virginica) of the Atlantic seaboard of the United States. These oysters live in estuaries, where the ambient salinity varies from place to place. Because their blood osmotic pressure matches the ambient osmotic pressure, their blood osmotic pressure also varies from place to place. The MSX parasite cannot survive in an oyster if its blood

Marine elasmobranch fish, although they have blood ion concentrations far lower than those of seawater, are slightly hyperosmotic to seawater because of high concentrations of two counteracting organic solutes, urea and trimethylamine oxide (TMAO). Unlike teleosts, therefore, elasmobranchs need not drink and need not incur an extra NaCl load to gain H2O from ingested seawater.

4E

is more dilute than about 400 mOsm. For the oysters, therefore, ambient waters with osmotic pressures lower than 400 mOsm are safe havens from the parasite. In the Chesapeake Bay (see Figure 27.4), serious spread of MSX occurs during droughts. When there is little rain, rivers bring less freshwater into the Bay, and the salinity rises above 400 mOsm in places where it is ordinarily lower. The oysters living in such places experience a rise in blood osmotic pressure and become vulnerable to the parasite.

Among animals that are osmoregulators, regulation is often limited to certain ranges of ambient osmotic pressure. Thus dif- ferent categories of regulators are recognized. In one common pattern, called hyper-isosmotic regulation, a species keeps its blood more concentrated than the environmental water at low environmental salinities, but allows its blood osmotic pressure to match the ambient osmotic pressure at higher salinities. Species that are predominantly freshwater animals but venture into brack- ish waters typically show this pattern, as do many coastal marine invertebrates (FigurE 28.11A). Animals exhibit hyper-isosmotic regulation when they possess mechanisms of hyperosmotic regula- tion but lack mechanisms of hyposmotic regulation.

(A) Hyper-isosmotic regulators

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FigurE 28.11 Types of osmotic regulation For each animal shown, blood osmotic pressure is plotted as a function of the osmotic pressure of the ambient water. Each dashed line is a line of equality between blood osmotic pressure and ambient osmotic pressure (an isosmotic line). (A) Three species of hyper-isosmotic regulators. Such regulation is typical of freshwater animals that enter brackish waters (e.g., the carp); it occurs also in many crabs of shores or estuaries (e.g., the blue crab, Callinectes sapidus, shown) and in some euryhaline annelids and amphipods (e.g., the amphipod Gammarus oceanicus, shown). (B) Four species of hyper-hyposmotic regulators. Such regu- lation occurs in many shore crabs (e.g., the fiddler crab, Uca pugilator, and the lined shore crab, Pachygrapsus crassipes), semiterrestrial crabs (e.g., the ghost crab, Ocypode cursor), coastal shrimps, and animals adapted to inland saline environments (e.g., the brine shrimp, Artemia salina), as well as euryhaline
and migratory fish. (After D’Orazio and Holliday 1985; Greenaway 1988; Hill and Wyse 1989; Kirschner 1991.)

A second major category of regulators consists of those that keep their blood more concentrated than the environmental water at low environmental salinities but more dilute than the envi- ronmental water at high environmental salinities. This pattern is called hyper-hyposmotic regulation and requires mechanisms of both hyperosmotic and hyposmotic regulation. It is observed in salmon, eels, and other migratory fish and in a variety of crustaceans (FigurE 28.11b).

Both osmoconforming and osmoregulating species occur among the crustaceans that live in the oceans and also in the marine annelids and some other related sets of marine animals. In these groups, euryhalinity and osmoregulatory ability tend to be correlated: The most euryhaline species are typically those that osmoregulate to a comparatively strong extent. Success in dilute waters in marine crustaceans, annelids, and other such groups, therefore, has been achieved by protecting the cells of the body from exposure to low blood osmotic pressures, in contrast to the oysters and mussels earlier discussed.

Migratory fish and other euryhaline fish are dramatic and scientifically important examples of hyper-hyposmotic regulators

The fish that migrate between freshwater and the oceans typically breed in one habitat and undergo much of their growth and matura- tion in the other. Some species—termed anadromous (“running upward”)—ascend rivers and streams from the oceans to breed; these fish include salmon and certain smelts, shad, and lampreys. Other species—termed catadromous (“running downward”)— grow in freshwater and descend to the oceans for breeding; they include the freshwater eels (genus Anguilla) of North America, Europe, and East Asia.

The migratory fish are superb osmoregulators. They function as hyperosmotic regulators when in freshwater and as hyposmotic regulators when in seawater, and they are so effective in both habitats that their blood osmotic pressure generally changes only a little between the two. Chinook salmon (Oncorhynchus tshawyts- cha), for example, have a plasma osmotic pressure averaging about 410 mOsm when in the ocean and about 360 mOsm when at their freshwater spawning grounds.

The mechanisms of regulation employed by migratory fish—and other euryhaline teleosts—in seawater and in freshwater are the same as those we earlier discussed for marine and freshwater tele-

0

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Water and Salt Physiology of Animals in Their Environments 759

Lined shore

Ghost crab Brine shrimp

crab

Fiddler crab

Osmotic pressure of blood (mOsm) Osmotic pressure of blood (mOsm)

5

760 Chapter 28
(A) Responses of gill proteins to transitions between

Some crustaceans that exhibit hyper-hyposmotic regulation, such as fiddler crabs (Uca; see Figure 28.11B), are known to display similar and equally dramatic shifts in their regulatory mechanisms as they move between salinities.

The migratory fish—and other euryhaline teleosts—have been and continue to be the most important of all fish for studies of the physiological regulation of water–salt rela- tions. They are studied intensely because their regulatory systems meet dramatic challenges and thus provide vivid insight into regulation in action.

One major objective of modern research on fish water–salt physiology is to understand the molecular mechanisms of successful transitions between freshwater and seawater. Studies of gill function provide a good illustration. In recent years, researchers have established that the gills of an indi- vidual fish undergo extensive molecular remodeling during transitions between freshwater and seawater—remodeling that leads to distinctive freshwater and seawater gill phenotypes. These phenotypic adjustments include critical changes in the cell morphology and the suites of ion-transport proteins in the mitochondria-rich cells (MRCs) that are so important for gill ion transport (see Box 28.1). Using monoclonal antibodies to assay defined cell-membrane proteins of the MRCs by use of immunocytochemistry, for example, researchers have studied the concentrations and types of Na+–K+-ATPase and NKCC (a Na–K–2Cl cotransporter) during freshwater-to-seawater transitions. Both of these ion-transport proteins are predicted—from knowledge of molecular transport mechanisms in teleost fish (see Boxes 5.2 and 28.3)—to increase in individuals transferred from freshwater to seawater. In studies of brown trout (Salmo trutta), quantitative changes in the proteins follow this prediction, as seen in FigurE 28.12: The proteins increase in the gill MRCs when trout are transferred to seawater and decrease when the fish are returned to freshwater. Studies of several other species confirm these results. Moreover, research on Atlantic salmon (Salmo salar) and some other species reveals that the molecular form of Na+–K+-ATPase also changes between freshwater and seawater, implying that the detailed function of the ATPase is modulated. Aquaporins constitute another area of molecular research. Recent studies (e.g., on eels, Anguilla japonica) indicate that aquaporins are upregulated in the intestinal epithelium following transfer to seawater—a response predicted to facilitate uptake of H O from ingested seawater.

complex endocrine controls of water–salt physiology. Years

ago, investigators discovered that hypophysectimized fish died when transferred to freshwater, but they could be rescued by the specific adenohypophysial hormone prolactin. Those experi- ments established that hormonal controls are of vital importance in water–salt physiology; prolactin, in particular, plays a key role in freshwater, not only in reducing the permeability of the gills to Na+ but also in augmenting urine flow. By now, several other hormones are known to be of central importance in water–salt physiology, including cortisol, growth hormone, insulin-like growth factor, and thyroid hormone.

freshwater and seawater

Transferred to seawater

Trout acclimating to seawater

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00 5 1060 65 700 Days

(B) Gill tissue in which NKCC is stained for identification

Acclimated to seawater

Acclimated to freshwater

Gill secondary lamella

Arrows point to some of the stained cells.

100 μm

Transferred to freshwater

Trout acclimating to freshwater

The abundance of each protein is expressed per unit of gill tissue
(in arbitrary units).

FigurE 28.12 Molecular phenotypic plasticity in gills of trout transferred between freshwater and seawater (A) Brown trout (Salmo trutta) that had been living in freshwater were transferred to sea- water on day 0. After living in seawater for 60 days (note the break in the
x axis), they were returned to freshwater. Abundances of Na+–K+-ATPase (α subunit) and NKCC (Na–K–2Cl cotransporter) were quantified by immuno- cytochemistry. (B) Images of gill secondary lamellae, from fish acclimated more than 60 days to seawater or freshwater, in which NKCC is visualized immunocytochemically by use of a monoclonal antibody specific to the protein, resulting in a red color (nuclei are stained blue). Note that staining is more intense in the seawater-acclimated gill. (A after Tipsmark et al. 2002; B courtesy of Christian Tipsmark, from Tipsmark et al. 2002.)

osts (see Figure 28.8). Thus, when the fish move from freshwater to seawater, they reverse the direction of active NaCl transport across their gills (inward transport in freshwater, outward in seawater); they greatly increase the rate at which they drink; they decrease their rate of urine production; and they switch from producing urine that is markedly hyposmotic to their blood plasma to producing urine that is approximately isosmotic to the plasma. In their intestinal epithelium, the activity of the NaCl-uptake mechanisms and the abundance of aquaporins also increase when they enter seawater.

2
A second major objective of research today is to clarify the

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Abundance of Na+–K+-ATPase

Abundance of NKCC

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living in its
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After North Sea mussels were switched to living at a salinity of 15 g/kg and Baltic Sea mussels were switched to living at 30 g/kg, ciliary activity in each group gradually came to resemble that originally seen in the other group.

KEY

• (B) 6 days after switch
• (C) 10 days after switch

(D) 30 days after switch

Hill Animal Physiology 4E
expSirneausesr iAosnsocciahtesanges in crustaceans than

Baltic Sea mussels living at Baltic Sea salinity: 15 g/kg

North Sea mussels living at North Sea salinity: 30 g/kg

Water and Salt Physiology of Animals in Their Environments 761

0 10 20 30 40 50 60 70 Salinity of the test water (g/kg)

FigurE 28.13 Acclimation of mussels to changed salinity
Blue mussels (Mytilus edulis) were collected from the North Sea, where the ordinary ambient salinity is about 30 g/kg, and from the Baltic Sea, where the ordinary ambient salinity is about 15 g/kg (for reference, open-ocean seawater has a salinity of about 35 g/kg). After initial testing (A), each group was switched to living in water of the opposite

A memorable and informative experiment was done recently by simply adding NaCl to the diets of rainbow trout kept in freshwater. One might conclude that the trout were fooled by the dietary salt because they responded by modifying their gill phenotype to that of seawater-acclimated fish! For example, they upregulated the genes coding for Na+–K+-ATPase and NKCC. The experiment indicates that salt exposure is a trigger for phenotypic remodeling.

fish

salinity and periodically retested (B–D).The aspect of their physiology studied was the activity of their gill cilia—important for pumping water through the body so food and O2 can be collected. Ciliary activity (y axis) was scored on a scale of 0 (low) to 3 (high) as a function of salin- ity (x axis). Blue mussels are of great importance in natural ecosystems and aquaculture. (After Theede 1965.)

Animals undergo change in all time frames in their relations to ambient salinity

The relations of animals to salinity can change in all the time frames we highlighted in Chapter 1 (see Table 1.2). Besides acute responses (the responses that individuals undergo soon after the salinity of their environment becomes altered), another time frame in which individuals respond is the chronic time frame (i.e., acclimation or acclimatization). The responses of trout shown in Figure 28.12 pro- vide one example of acclimation. Another instructive example comes from studies of groups of blue mussels (Mytilus edulis) collected from the North Sea—where the salinity of the ambient water was 30 g/ kg—and from the brackish Baltic Sea—where the salinity was 15 g/kg. FigurE 28.13A shows the ranges of salinities at which the two groups of mussels were able to maintain ciliary activity (rated on a scale of 0–3 on the y axis) at the time of collection. The groups were then switched in the salinities at which they lived. As each group acclimated to its new salinity (FigurE 28.13b–d), the range of salinities over which it could maintain ciliary activity gradually shifted. After 30 days, the North Sea animals living at a salinity of 15 g/kg displayed normal ciliary activity over approximately the same salinity range as originally seen in the Baltic Sea animals; the Baltic Sea animals living at a salinity of 30 g/kg also acclimated, coming to resemble the original North Sea animals. These results reveal acclimation in action and suggest that the original differ- ence between the North Sea and Baltic Sea mussels was largely a consequence of acclimation (individual phenotypic plasticity).

genomic studies point to greater gene-

Figure 28.13 12-23-15

The meta-analysis by Wilson and his colleagues, based on data gathered in many labs on many species exposed to a variety of salinity changes, did identify a general pattern: Crustaceans tend to exhibit about threefold greater changes in expression of the genes coding for Na+–K+-ATPase and NKCC than teleost fish do. This genomic insight will need now to be assessed to determine the causes and consequences of this pattern of gene expression at higher levels of organization, such as in tissues and organs (see Figure 3.8).

Over the past 10 years many studies have used newly available ge- nomic methods to measure altered expression of key ion-transport genes in the gills after animals are switched from one environment to another, such as from seawater to freshwater. Alan E. Wilson and colleagues recently completed a meta-analysis of almost 60 such studies. The goal of meta-analysis is to use statistical methods to analyze numerous sets of data simultaneously, to identify patterns that the data sets collectively indicate are present.

Ciliary activity on a scale of 0–3

Mussels from North Sea

Mussels from Baltic Sea

762 Chapter 28

Evidence also suggests that populations of a species can evolve differences in their water–salt physiology when living in different environments over multiple generations. An intriguing illustration is provided by populations of lampreys (Petromyzon marinus) that have become landlocked in North American freshwater lakes. Lampreys, which are anadromous like salmon, ordinarily migrate to the ocean when they are young adults. The populations that are landlocked in lakes have had no experience with the sea for many generations, however. Adults from some landlocked populations exhibit osmoregulatory difficulties when they are placed in water that is only half the salinity of seawater. However, adults from migratory populations—when tested—can osmoregulate at the full salinity of seawater even before they have migrated and had actual experience with salty waters. These observations strongly suggest genetic divergence between the landlocked and migratory populations.

Many animals are also known to undergo developmental changes in their water–salt physiology. Recall, for example, the changes we have discussed in the locations of mitochondria-rich cells (chloride cells) in developing fish (see Figure 4.6).

only to its mouth. The cocoon acts as a barrier to evaporative water loss. The fish then enters a state of metabolic depression (see page 208): Its metabolic rate ultimately drops to about 10% of the ordi- nary resting level. This hypometabolism reduces its rate of respiratory water loss, and also its rate of use of stored energy. The lungfish’s kidneys virtually stop making urine—keeping water in the body but compelling wastes to accumulate. The lungfish switches from producing ammonia as its principal nitrogenous end product to producing urea, a far less toxic compound (see Box 28.4), and urea may accumulate in its blood to levels approaching those of marine elasmobranch fish. The lungfish can survive in this dormant condi- tion for more than a year!

Many other freshwater animals burrow into the substrate—often encasing themselves in mucous coverings—and enter a resting condition during times of drought. Included are some leeches, snails, water mites, and amphibians.

Anhydrobiosis—“remaining alive without water”—refers to survival while dried as fully as possible by desiccation in air. It represents the extreme in animal desiccation tolerance (bOx 28.5). A wide diversity of small animals from freshwater, saline, and ter- restrial habitats are capable of anhydrobiosis, during which they become inert and as dry as paper or any other air-dried organic matter: They are so dry that they become like dust. In anhydrobiosis these animals are often tolerant of a variety of environmental extremes, not just extreme dryness, and often they can endure the air-dried state for many years. They frequently blow about in the wind, springing magically back to an animated life when they land in water. Biologists have long recognized two advantages of anhydrobiosis: It permits survival without water and can aid dispersal. A striking experiment on anhydrobiotic bdelloid rotifers has recently revealed a new potential advantage: escape from pathogens. Populations of rotifers exposed to a potentially lethal fungus were subjected to desiccation for various lengths of time. If desiccation continued for 4–5 weeks, 80%–90% of the populations were fungus-free after rehydration and the rotifers lived—because the rotifers tolerated desiccation longer than their fungal pathogen (FigurE 28.14).

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FigurE 28.14 A long period of anhydrobiosis enhances sur- vival of bdelloid rotifer populations because, during desic- cation, the rotifers outlast their fungal pathogen Populations of the bdelloid rotifer Habrotrocha elusa were seeded with conidia of the fungal parasite Rotiferophthora angustispora and desiccated.The graph shows the percentage of rotifer populations killed by fungal growth following rehydration, as a function of the time until the popu- lations were rehydrated. (After Wilson and Sherman 2010.)

responses to drying of the Habitat in Aquatic Animals

Residents of puddles, small ponds, intermittent streams, and the like are often confronted with drying of their habitat. The lungfish (dipnoan fish), which have lungs and fleshy fins, are of particular interest in this regard because of their relatively close relation to the fish that gave rise to amphibians. All species of lungfish occur in transient bodies of freshwater. If the habitat dries out, an African lungfish (Protopterus aethiopicus) digs a chamber in the bed of the lake or stream where it has been living; in the chamber, the fish curls up and secretes mucus that hardens into a cocoon opening

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Percentage of rotifer
populations killed by fungus

As we now turn to animal life on land, we retuHrinll toAnciomnasl iPdheyrsionlgogy 4E Sinauer Associates

animals in their active, alert states, going about their daily lives.

They will be our focus except for occasional brief discussions of dormancy.

As emphasized already, animal life originated and spent much of its early evolutionary history in water. The earliest animals that ventured to spend time on land, to consume terrestrial organisms as food, and ultimately, to develop on land were able to escape competitors and predators in their primordial aquatic habitat. For this reason, positive selective pressure for terrestriality must have been great. However, early animal life was adapted to living in an abundance of water. Evaporative losses of water on land posed a physiological threat of enormous importance for all stages of the life cycle.

We will focus on water in our discussion of animals on land. Although terrestrial animals sometimes face problems of salt balance, water balance usually represents their most pressing challenge in the realm of water–salt physiology.

The distinction between humidic and xeric animals provides a useful organizing principle for the study of water relations in ter- restrial animals. The humidic animals are those that, although

Others live in leaf litter or under logs or rocks. The majority of frogs and toads stay in or near bodies of water, and when they venture away from water, they remain in protected microenvironments, such as the tall grass frequented by leopard frogs. Some humidic animals, such as most amphibians and all terrestrial crabs, still resemble their aquatic progenitors in that they require standing water to breed.

The major groups of xeric animals are the mammals, birds, reptiles other than birds, insects, and arachnids (e.g., spiders and ticks). Although xeric animals often seek protected, humid micro- environments, they are not stringently tied to such environments, as humidic animals are. The xeric animals can live successfully in the open air, and many of them—as they go about their daily lives—routinely expose themselves to the full drying power of

15 The term xeric has a standardized meaning and is widely used. However, there is no standardized term to describe the animals restricted to moist habitats; although we use humidic, alternative terms are used in other books and articles. The term mesic is sometimes applied to animals intermediate between those that are xeric and those that are humidic.

Water and Salt Physiology of Animals in Their Environments 763

Animals on land: Fundamental

Physiological Principles

they live on land, are restricted to humid, water-rich microenviron- ments. The xeric animals are those that are capable of living in dry, water-poor environments.15

The humidic animals include earthworms, slugs, centipedes,

most amphibians, and most terrestrial crabs. Some live underground.

Box Figure 28.05 12-23-15

764 Chapter 28

the terrestrial environment. Some thrive in deserts and other equally dry environments, such as grain stores.

In many ways, the physiological difference between humidic and xeric animals is a distinction in how rapidly they get into trouble by loss of water in desiccating environments. The humidic animals dehydrate rapidly in dry environments. They therefore cannot remain long in such places, as xeric animals can.

A low integumentary permeability to water is a key to reducing evaporative water loss on land

Evaporation is one of the chief modes by which terrestrial animals lose water. In this section we begin our analysis of the physiological principles of living on land by discussing evaporation across the integument of the body. As discussed in Chapter 27 (see Equation 27.1), the rate of evaporation through an animal’s integument de- pends on the difference in water vapor pressure between the animal’s body fluids and the air, and it depends on the permeability of the in- tegument to water (K in Equation 27.1).

A high integumentary permeability to water ranks as one of the most important specific characteristics that restrict humidic animals to their protective microhabitats. The skin of an earthworm, the skin of most amphibians, and most of the fleshy surfaces of a snail or slug, for example, have high permeabilities and provide little barrier to water loss: These animals often lose water through their integuments at rates that are 50%–100% as great as rates of evaporation from open dishes of water of equivalent surface area! With such a high integumentary permeability, a humidic animal can restrict its integumentary rate of evaporation only by limit- ing the difference in water vapor pressure that exists across its integument. From the viewpoint of physics, this explains why humidic animals are tied to humid habitats, where the air has a high water vapor pressure.

The xeric animals have integuments with a low permeability to water. Indeed, the evolution of a low integumentary permeability to water is one of the most important steps toward a xeric existence.

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FigurE 28.15 The rate of evaporative water loss of insects often starts to increase abruptly at a transition tempera- ture The graph shows how the rate of evaporative water loss of dead African migratory locusts (Locusta migratoria) increases as temperature increases.The rate of water loss is expressed“per mm Hg,” referring to the difference (expressed in millimeters of mercury) between the actual water vapor pressure of the air during the mea- surements and the saturation vapor pressure at the various tempera- tures. Measured and expressed in this way, changes in the rate of evaporative water loss reflect changes in the water permeability of the integument. (After Loveridge 1968.)

and centipedes either lack the micrometer-thin epicuticular lipid layer or possess lipids of different types than insects. They are far more humidic than insects because of these microscopic differences.

Lipids reorganize and undergo phase alterations as their tem- perature changes, as evidenced by observing kitchen lipids such as butter. Researchers have long known that if the temperature of an insect (or arachnid) is gradually raised, water permeability increases just slightly up to a certain temperature—called the transition temperature—and then increases dramatically (FigurE 28.15). The marked increase in permeability at the transition tem- perature is a consequence of lipid melting. Although the transition temperature is often so high that it would not be experienced by individuals in nature, this is not always the case. The cockroach Periplaneta americana (a common household pest), for example, experiences a marked increase in permeability starting at 25°C– 30°C and thus might naturally encounter temperatures high enough to disrupt its protection against water loss. Temperature effects within the skin of vertebrates are far more complex because of changes in blood flow and other processes, but careful stud- ies reveal that in at least some cases, a rise in epidermal tissue temperature decreases the effectiveness of the cutaneous lipid layer as a water barrier.

Within sets of related xeric animals, the chemical composition of the lipid layer can vary widely. Because of these lipid composition differences and also sometimes because of structural differences or differences in amounts of lipids, the lipid layer can be far more effective as a water barrier in some species than in other related species. TAblE 28.6 shows, for example, that the resistance of skin to water loss—measured in a standardized way—varies widely within groups of xeric vertebrates as well as between such groups.

In all the major xeric groups—vertebrate and invertebrate—micro- Hill Animal Physiology 4E

scopically thin layers of lipids are responsible for low integumentary Sinauer Associates

water permeability. In mammals, birds,Faignudren2o8n.1a5via1n2-r2e3p-t1i5les (e.g., lizards and snakes), the lipid layers are structurally heterogeneous, lamellar complexes of lipids and keratin, less than 10 micrometers (μm) thick, located in the stratum corneum, the outermost layer of the epidermis of the skin. The principal lipids present are ceramides, cholesterol, and free fatty acids. Mammals, birds, and nonavian reptiles differ in histological details of the lipid layers, and they evidently evolved their lipid layers independently. In insects and arachnids, the lipids responsible for low integumentary perme- ability—such as long-chain hydrocarbons and wax esters—are contained in the outermost layer of the exoskeleton. This layer, termed the epicuticle, is only 1–2 μm thick.

Because virtually all the resistance to water loss across the integument of xeric animals resides in microscopically thin lipid layers, the physical toughness of the integument is not an index of resistance to water loss. A common misconception, for example, is that the scales of lizards and snakes block water loss; the real block is the lipid layer, a microscopic property of just the stratum corneum. Many millipedes and centipedes have sturdy exoskeletons that seem just as tough as those of insects, yet their exoskeletons are far more permeable to water than those of insects. Such millipedes

0 10 20 30 40 50 60 Air temperature (°C)

transition temperature— ut 40°C in this case—is as as 25°C in some insects

as high as 55°C in others.

Rate of water loss (mg H2O/g•h•mm Hg)

Source: Lillywhite 2006.
a Resistance is calculated as the inverse of conductance. Conductance

is the rate of water loss across the skin—expressed as grams H2O per cm2 of skin per second—divided by the driving force, which in this case is the difference in water activity across the skin—expressed in water density units of grams H2O per cm3 of air. For a units analysis, one divides g/cm2•s by g/cm3.Thus the units of conductance are cm/s, and the units of resistance, the inverse, are s/cm.

The lipid composition of the water barrier may even differ among populations of a single species and give rise to significant differences between populations in their physiology of water balance (FigurE 28.16). At still another level of organization, cases are known—as in certain desert larks—in which the lipid composition of individuals changes as a consequence of acclimation to different environments.

2 1.5

1

0.5

0.2 0.3
Body weight (g) on log scale

FigurE 28.16 differentiation between populations of one species in lipid-mediated protection against evaporative water loss The graph shows the rate of evaporative water loss at 25°C as a function of body weight in live grasshoppers of a single species (Melanoplus sanguinipes) from two geographically separate populations in northern and southern California.According to current evidence, these populations exhibit genetically controlled differences in the lipid composition of their epicuticular layer.These differences contribute to the greater resistance of the southern animals to water loss. In each population, the rate of water loss is an allometric func- tion of body weight, so the relation plots as a straight line on log–log coordinates (see Appendix F). (After Gibbs 1998.)

respiratory evaporative water loss depends on the function of the breathing organs and the rate of metabolism

Some groups of humidic animals have respiratory surfaces that are directly exposed to the air. Earthworms, some isopods, and some amphibians, for instance, breathe substantially or entirely across their general integument (skin or exoskeleton). This arrangement is a disadvantage from the viewpoint of evaporative water loss because movement of air across exposed respiratory surfaces can greatly exceed that necessary for exchange of O2 and CO2, so water loss can be much greater than the minimum required for respira- tory gas exchange.

Most terrestrial animals have evolved invaginated respiratory structures (see Figure 1.20). In the xeric groups, breathing is carried out exclusively by such invaginated structures, and the general integument is virtually impermeable to O2 and CO2. The enormous advantage of this arrangement is that access of air to the thin, moist respiratory membranes can be closely controlled and thus limited to the levels required for exchange of O2 and CO2. The mammals, birds, and other reptiles control access of air to their lungs by regulating their breathing movements. Insects close and open the spiracles of their tracheal system (see Figure 23.31).

THE EFFECT OF TEMPErATurE ON THE WATEr vAPOr CONTENT OF Air A physical law of great consequence for warm-bodied air breathers is that when air is saturated with water vapor, its content of water per unit of volume approximately doubles with every 11°C increase in temperature (see Table 27.2).16 Changes of air tem- perature can thus exert a strong influence on the amount of water carried by air movement.

When a mammal or bird inhales air into its lungs, the temperature of the air is raised to deep-body temperature, and the air becomes saturated with water vapor at its new, elevated temperature. As a consequence, depending on conditions, a substantial amount of water may be added to the inhaled air. For example, consider a mammal that inhales saturated air at 20°C. Such air contains about 17 mg H2O/L (see Table 27.2). By the time the air reaches the lungs, it is saturated at 37°C, and it therefore contains 44 mg H2O/L (see Table 27.2). Thus, even though the air is saturated to begin with, it contains 27 mg/L more water once it is in the lungs, all of this added water being drawn from the animal’s body. If the air is then exhaled without modification, it carries all the added water away into the environment.

WATEr CONSErvATiON by COOliNg OF ExHAlANT Air

When a mammal or bird exhales, the exhaled air usually is satu- rated with water vapor. However, in many mammals and birds, if the air is exhaled by way of the nasal passages, the temperature of the air is reduced before the air leaves the nostrils, a process that lowers the saturation water vapor pressure and therefore reduces the amount of water the air carries away.17 Suppose the mam- mal we discussed in the preceding paragraph were to reduce the temperature of air from its lungs to 25°C before exhaling the air. On leaving the body, the air would then contain 23 mg H2O/L

16 As seen in Table 27.2, the saturation water vapor pressure of air increases with temperature, and the amount of water vapor per unit of volume increases in parallel.

17 Cooling of nasal exhalant air also takes place in some lizards when they are maintaining high, behaviorally regulated body temperatures.

0.4 0.5

Water and Salt Physiology of Animals in Their Environments 765

Northern population Southern population

Rate of evaporative water loss (mg H2O/h) on log scale

766 Chapter 28

(see Table 27.2). The air would still carry away some body water (it entered the body with 17 mg H2O/L). However, the reduction in the temperature of the exhalant air would cause 78% of the water added during inhalation to be recovered before exhalation.

In those mammals and birds that reduce the temperature of air before it is exhaled, the air is cooled by a countercurrent mechanism in the nasal passages. To understand the process, let’s first look in more detail at what happens during inhalation, using our example of a mammal breathing 20°C air. As inhaled ambient air travels up the nasal passages, it is progressively warmed to about 37°C, and it takes up water vapor as its temperature is elevated. The heat that warms the air and the latent heat of vaporization for the added water vapor are drawn from the walls of the nasal passages.18 Thus the walls of the nasal passages are cooled by the process of inhalation. The outer ends of the nasal passages are typically cooled most, and the inner ends least. During the ensuing exhalation, air coming up from the lungs arrives at the interior ends of the nasal passages at a temperature of 37°C and saturated. However, as the air moves down through the nasal passages toward the nostrils, it encounters the increasingly cooler surfaces created by the previous inhalation. Thus the air being exhaled is cooled as it travels toward the nostrils. This cooling lowers the saturation water vapor content of the air (see Table 27.2), causing water to condense out of the air onto the nasal-passage walls. The overall process is considered a countercur- rent process because it depends on flow of air in opposite directions.

If the cooling of nasal exhalant air seems unfamiliar, it may be because only a small degree of cooling occurs in humans. In contrast, the cooling of nasal exhalant air in small mammals is dramatic, as illustrated by the data plotted as black dots in Figure 28.17; a small mammal that inhales air at 20°C and warms it to 37°C in its lungs might well exhale the air at 22°C–23°C.

a useFul model oF respiratory evaporative water loss

An insight-promoting way to think about the rate of respiratory evaporative water loss is to recognize that it depends on (1) an animal’s rate of O2 consumption and (2) the amount of water lost per unit of O2 the animal consumes:19

Deep-body temperature is 37°C in the mammals and 39°C in the birds. The air these animals inhale is warmed to deep-body temperature in the lungs, but it is cooled to be closer to ambient temperature than to deep-body temperature by the time it is exhaled. A large water saving results.

30

25

20

15

Rate of water loss = rate of O2 consumption

× water loss per unit of O 2

(28.1)

consumed

10
10 15 20 25 30

Ambient air temperature (°C)

Figure 28.17 the temperature of air exhaled from the nos- trils as a function of ambient air temperature in mammals and birds The black dots are data for individual small mam-
mals (e.g., shrews, bats, mice, squirrels, and rabbits); 18 species are included.The solid lines are average results for mongrel domestic dogs (blue) and cactus wrens (Campylorhynchus brunneicapillus) (red).The dashed line is a line of equality between the temperature of exhaled air and the ambient air temperature (an isothermal line). (From Hill and Wyse 1989.)

The amount of water lost per unit of O2 consumed is affected by several factors. One is the temperature of the exhaled air we just discussed (lower exhalant temperatures mean lower water loss). Another is the efficiency of the breathing organs in removing O2 from inhaled air.

an animal’s total rate of evaporative water loss depends on its body size and phylogenetic group

If we sum an animal’s rates of integumentary and respiratory water lossH,ilwl eAgneimtatlhPehyasnioilmogayl4’sEtotal rate of evaporative water loss (EWL).

There are two reasons why small-bodied species tend to have relatively high weight-specific rates of EWL. First, small animals tend to have a greater body surface area per unit of weight than related large animals (see Equation 7.6); therefore they tend to have relatively high weight-specific rates of integumentary water loss. Second, small animals tend to have a higher metabolic rate per unit of body weight than related large animals (see Figure 7.9); therefore they tend to have relatively high weight-specific rates of respiratory water loss (see Equation 28.1).

There are also consistent differences among phylogenetic groups in their total rates of EWL, as we have stressed. Animals in humidic phylogenetic groups, such as amphibians, have highly permeable integuments and other properties that give them high total rates

The principal insight to gain from this equation is that an animal’s rate of metabolism is a major determinant of its rate of evaporative water loss. Mammals and birds, as we stressed in Chapters 7 and 10 (see Figure 7.9), typically have rates of O2 consumption that are far higher than those of lizards, snakes, or other nonavian reptiles of similar body size. Mammals and birds therefore tend to have high rates of respiratory evaporative water loss by comparison with lizards, snakes, and other nonavian reptiles.20

18 The nasal passages are not simple tubes. Their walls often consist of elaborate surface-enhancing structures (turbinates) over which air flows, as can be seen in the insets in Figure 10.37.

19 If the rate of O2 consumption is measured in milliliters of O2 per hour (mL O2/h), and if the water loss per unit of O2 consumed is measured as mg H2O/mL O2, when these two factors are multiplied, the units of the result will be mg H2O/h. That is, the result will be a measure of the rate of water loss in milligrams per hour.

20 A compensation is that animals with relatively high rates of O2 consumption also have relatively high rates of metabolic water production (e.g., see Figure 28.18).

Figure 28.17 12-23-15

Sinauer Associates

In this case, the animal inhaled air at 24oC and exhaled it only slightly warmer, about 26oC.

Each black dot represents a single small mammal.

Within sets of phylogenetically related species, the broad statisti-

cal trend is for the total rate of EWL measured under particular conditions to vary allometrically with body size. This important pattern is illustrated in Figure 28.18 using birds as examples. Small-bodied species tend to have higher weight-specific rates of EWL than related large-bodied species.

Dog

Wren

Isothermal line

Temperature of exhaled air (°C)

al Physiology 4E ssociates
.18 12-23-15

Semiterrestrial crabs

Isopods (sowbugs, pillbugs)

Insects

Vertebrates (16–39 g)

Ghost crab (44 g) Fiddler crab (5 g)

Intertidal isopod (0.9 g) Pillbug (0.2 g)

Migratory locust (1.9 g) Tsetse fly (0.03 g)

Semiaquatic frog Desert spadefoot toad

Desert iguana lizard Sand lizard

Desert kangaroo rat Lab mouse

House finch Brown towhee

40

30

20

10

00 40

FigurE 28.18 Within a phylogenetic group, the total rate of evaporative water loss is an allometric function
of body size This trend is illustrated here (black line and black dots) using data for birds resting at 23°C–25°C in relatively dry air. Evaporative water loss is expressed in weight- specific units (the expression percentage of body weight per day is equivalent to mg H2O lost per 100 mg of body weight per day).The black line is fitted statistically to the individual data points (black dots).The red area delimits the gross rates at which birds of various body sizes are expected to produce metabolic wa- ter (expressed as percentage of body weight per day). (From Hill and Wyse 1989.)

of EWL in comparison with similar-sized animals in xeric groups. Some of these differences in total rates of EWL are brought to light in FigurE 28.19. All the vertebrates in the figure are about the same in body size; therefore, comparing them brings out differences among the phylogenetic groups. Lizards (and other nonavian reptiles) have very low total rates of EWL compared with amphibians because the lizards have low-permeability integuments (see Table 28.6) and enclosed breathing systems. Mammals and birds share these basic properties of lizards, but they have higher total rates of EWL than

lizards (and other nonavian reptiles). Why? The principal reason is that they have far higher metabolic rates than lizards. The two species of semiterrestrial crabs in Figure 28.19 are similar in size to the vertebrates; semiterrestrial crabs, as can be seen, are similar to amphibians in their rates of EWL and are humidic. The isopods and insects in the figure are tiny compared with the other animals shown. The isopods combine the disadvantages of small size and poor defenses against EWL. Their total weight-specific rates of EWL are staggering. One can only marvel at the insects, especially the

FigurE 28.19 The total rate of evaporative water loss varies greatly among different types of vertebrates and arthro- pods All the animals shown were studied in dry or relatively dry air at 25°C–32°C.The vertebrates (red) are all similar in body size (16–39 g). Body weights are given for the individual arthropod species (blue). Evaporative water loss is expressed in weight-specific units (the expres- sion percentage of body weight per hour is equivalent to mg H2O

lost per 100 mg of body weight per hour). Species, listed from top to bottom: semiterrestrial crabs, Ocypode quadrata and Uca annulipes; isopods, Ligia oceanica and Armadillidium vulgare; insects, Locusta migratoria and Glossina morsitans; amphibians, Rana temporaria and Scaphiopus couchii; lizards, Dipsosaurus dorsalis and Uma notata; mammals, Dipodomys merriami and Mus domesticus; birds, Carpoda- cus mexicanus and Pipilo fuscus. (After Hill and Wyse 1989.)

80 120
Body weight (g)

160

200 4800 84,000

Water and Salt Physiology of Animals in Their Environments 767

0 0.5
Weight-specific total rate of evaporative water loss (percentage of body weight per hour)

1.0

1.5 2.0

2.5 3.0

Weight-specific total rate of evaporative water loss (percentage body weight/day)

m A

8

768 Chapter 28

tsetse flies (weighing 0.03 g), which have such excellent defenses against water loss that they have relatively low total rates of EWL per unit of weight, despite being very small.

Excretory water loss depends on the concentrating ability of the excretory organs and the amount of solute that needs to be excreted

In addition to evaporative water loss, excretion in urine is a second major way that terrestrial animals lose water. Like aquatic animals, terrestrial ones modulate the concentration, composition, and volume of their urine to serve changing requirements for osmotic, ionic, and volume regulation. We humans, for example, when de- hydrated, produce a limited volume of urine that is hyperosmotic to our blood plasma (our osmotic U/P ratio can reach 4, meaning that our urine osmotic pressure can be four times the osmotic pres- sure of our blood plasma). However, after we have consumed large amounts of water, we void a copious urine that is hyposmotic to our blood plasma (our osmotic U/P ratio can be as low as 0.1–0.2). While recognizing the fundamental regulatory role of urine excretion, a key question in the study of water balance in terrestrial animals is how effectively the animals can minimize their urinary water losses. There are two basic ways to reduce the amount of water lost in urine. One is to concentrate the urine, thereby decreasing the amount of water required to excrete a given amount of solute. The second is to reduce the amount of solute excreted in the urine.

uriNE-CONCENTrATiNg AbiliTy Most of the humidic terres-

trial animals, such as earthworms and amphibians, are unable to

raise the osmotic pressure of their urine above that of their blood

plasma. Lizards, snakes, and turtles, although xeric, are also gener-

ally incapable of making urine that is hyperosmotic to their blood

plasma. By contrast, three of the major xeric groups—insects,

mammals, and birds—have evolved the ability to make hyperos-

motic urine: urine more concentrated than their blood plasma. This

unusual capability evolved independently in the three groups—a

fact that emphasizes the selective advantages of being able to make

10,000 5000

1000 500

100

10 g

100 g
Body weight on log scale

100 kg

1000 kg

concentrated urine in animals confronted with desiccation stress.

FigurE 28.20 Maximum urine-concentrating ability in mam- mals: The maximum concentration is in part a function of body size Each dot represents one species. Data for 146 species of mammals are included.The species chosen for labeling were delib- erately selected to represent extremes. Because the overall relation is allometric, it plots as a straight line on log–log coordinates (see Appen- dix F). Because all mammals have approximately the same plasma os- motic pressure, a plot of the maximum osmotic U/P ratio as a function of body size would resemble this plot. (After Beuchat 1990.)

body size tend to represent species that confront relatively severe threats of desiccation or high dietary salt loads.

In birds, the most evident point to stress is that, as a group, birds are far inferior to mammals in their abilities to concentrate their urine. Although osmotic U/P ratios approaching 6 have been claimed in a few species of birds, the maximum U/P ratio ranges from 1.5 to 2.5 in most species, including many that live in arid regions.

Some terrestrial lizards and birds (e.g., ostriches and roadrun-

ners) have cranial salt glands, which assist with secreting Na+, K+,

–
and Cl . The secretions of the salt glands have higher ion concentra-

tions than the urine in these animals. Thus the salt glands play an important role in decreasing the water expended to void excess ions.

rEduCiNg THE AMOuNT OF diSSOlvEd MATTEr ExCrETEd iN THE uriNE Waste nitrogen from the catabolism of proteins is usually excreted in the urine by terrestrial animals.21 One way to reduce the water demands of excretion is to incorporate the waste nitrogen into chemical compounds that are virtually insoluble—or poorly soluble—in water, thereby reducing the amount of material voided in solution. It is a testimony to the advantages of this type of nitrogen excretion that it has evolved independently many times: Insects, arachnids, some terrestrial snails, a few xeric frogs, birds, and other reptiles all produce poorly soluble nitrogenous wastes. The principal poorly soluble compounds employed are uric acid,

21 See Chapter 29 (page 809) for a more thorough discussion of nitrogen excretion.

Let’s look at the three in more detail. Hill Animal Physiology 4E Sinauer Associates

In terrestrial insects, maximum osmotic U/P ratios of 2–4 have

been observed in certain blowflies, desert locusts, and stick insects. Mealworms (Tenebrio), which live in dry grain stores, can produce urine with a U/P ratio of 8.

Mammals hold the world records for urine-concentrating ability. Although mammals display an enormous range of urine- concentrating abilities, the U/P ratios achieved by many species are well above those seen in any other animals. To illustrate both of these points, the maximum reported osmotic U/P ratio is about 3 for muskrats, 4 for humans, 8 for dromedary camels, 9 for labora- tory rats, 14 for Merriam’s kangaroo rats and Mongolian gerbils (Meriones), and—at the highest extreme—about 26 for certain species of Australian desert hopping mice (e.g., Notomys alexis)! A significant and unexpected feature of mammalian kidney function is that the maximum concentrating ability tends to decrease with increasing body size, as seen in FigurE 28.20. Much of the scatter in Figure 28.20 correlates with habitat; the highest values at a given

Figure 28.20 12-23-15

1000 g

10 kg

Australian hopping mouse

Kangaroo rat
Kirk’s dik-dik

Dromedary camel

Muskrat

Beaver

Maximum urine osmotic pressure (milliosmol/kg) on log scale

urate salts, allantoin, and guanine. Urate salts such as sodium and potassium urate—excreted by birds, other reptiles, and some ad- ditional groups—have the advantage that they carry away not only nitrogen but also inorganic cations in precipitated form.

By no means do all terrestrial animals exclude waste nitrogen from being excreted in solution, however. Many of the humidic animals—including earthworms, isopods, and most amphib- ians—excrete nitrogen principally as urea or ammonia, both of which are highly soluble. Mammals, paradoxically, also produce mostly urea (see Boxes 28.4 and 29.4).

If highly soluble nitrogenous wastes are voided in the urine, they demand water for their excretion. However, some of the animal groups that produce highly soluble wastes have evolved means of reducing the water demands. Mammals, for example, possess world-record abilities to concentrate urea in their urine (see Box 29.4). Some isopods, snails, and land crabs void ammonia as a gas.

terrestrial animals sometimes enter dormancy or tolerate wide departures from homeostasis to cope with water stress

Many terrestrial animals, both humidic and xeric, enter dormancy— often called estivation—in response to immediate or predictable water stress. Mammals and birds that enter water-related dor- mancy—including desert ground squirrels and poorwills—often undergo metabolic depression and become hypothermic (see page 274). Metabolism is depressed during dormancy in poikilotherms as well.22 Metabolic depression has several advantages. First, an animal in metabolic depression can live on body fat or other stored foods for a long time, and thus it can remain continuously in a protective microenvironment. Second, the animal’s requirement for O2 is reduced, thereby reducing its respiratory evaporation. Finally, nitrogenous and other wastes are produced at a low rate, which may be vital in permitting protracted existence with little or no urine output.

In addition to dormancy, another “change of status” that animals—particularly poikilotherms—commonly undergo during water stress is to permit large changes to occur in their body-fluid volume or composition while they continue to be active. Species

10

1

0.1

0.01

0.001 0.1 g

On average, if 100-g animals living in their natural habitats are compared, a nonavian reptile turns over 1.8 mL

that live in places where they are prone to dehydration are often

Figure 28.21 terrestrial vertebrates living freely in their natural habitats: their total daily rates of water turnover in relation to body size The water turnover is the amount of water lost and gained per day when animals are in water balance. It is plot- ted here in weight-specific terms (mL/g).The weight-specific version of Equation 28.2 is T/W = aW (b – 1). Each line is a plot of this equation. Because the relation for each group is allometric, it plots as a straight line on log–log coordinates. (After Nagy and Peterson 1988.)

gained per day) of terrestrial animals when they are living freely in their natural habitats. The rate of water turnover is typically mea- sured by use of isotopically labeled water (e.g., heavy water). An animal living in the wild is trapped, injected with a known amount of labeled water, and turned loose. Days later, it is recaptured, and from the amount of labeled water remaining in its body, its rate of water turnover while free can be calculated. For an animal in water balance, water lost by evaporation, urination, and other processes each day is replaced by gains of water. The rate of water turnover measures the rates of these mutually balancing processes. A high rate of water turnover means that an animal loses and replaces a lot of water per day. Life can be precarious for such an animal because if an imbalance develops, it can lead rapidly to a crisis.

Figure 28.21 shows the general water-turnover patterns of vertebrates living in the wild. Note that mammals and birds of any given body size turn over far more water per day than lizards, snakes, or other nonavian reptiles of the same size. Within any one phylogenetic group, as might be guessed from what we have said before in this chapter, the total rate of water turnover, T, tends to be an allometric function23 of body weight (W):

T = aWb (28.2)

The exponent b is typically 0.6–0.8. This means that if we consider the relation between the weight-specific rate of water turnover (T/W) and weight, the exponent (b – 1) is negative: –0.2 to –0.4. There-

Hill Animal Physiology 4E especially tolerant to such changes. Tortoises in the Mohave Des-

ert, for example, sometimes drop in body weight by 40%—and

beetles in East African savannas sometimes lose 80% of their body water—because of dehydration during droughts or dry seasons. When Claude Bernard spoke of the internal environment in his groundbreaking studies that led to the concept of homeostasis, he was referring to the body fluids (see page 13). The ability to remain active and functional despite profound alteration of the body fluids has been termed anhomeostasis and can be a key to existence during water stress.

the total rates of water turnover of free-living terrestrial animals follow allometric patterns

A logical way to conclude our introductory discussion of animals on land is to focus on the total rates of water turnover (water lost and

22 Suspension of metabolism during anhydrobiosis is discussed in Box 28.5.

23 See Appendix F for a discussion of allometric functions.

Sinauer Associates

Figure 28.21 12-23-15

Water and Salt Physiology of Animals in Their Environments 769

1 g

10 g
Body weight on log scale

10 kg

100 kg

1000 kg

100 g

1 kg

Marsupials

per day, a eutherian mammal turns over 14 mL per day, and a bird turns over 33 mL.

Birds

Eutherian mammals

Reptiles other than birds

Weight-specific amount of water (mL/g) lost and gained per day on log scale

770 Chapter 28

fore, as seen in Figure 28.21, the rate of water turnover per gram of body weight decreases as animals get bigger: Big species within a particular phylogenetic group tend to turn over a smaller fraction (lower percentage) of their total water per day than little ones.

of habitats has depended to a substantial extent on the evolution of protective behaviors and advantageous patterns of seasonality.

Most species of amphibians, including most that live in deserts, share several attributes that significantly limit their physiological capacity to restrain water losses. First and foremost, they have an integument that poses little barrier to evaporative water loss. Second, they incorporate waste nitrogen mostly into urea, a highly soluble compound requiring considerable amounts of water for its excretion. Moreover, although amphibians are notably adept at simply shutting off urine outflow when faced with dehydration, they are unable, when they do excrete urine, to produce a urine any more concentrated in total solutes than their blood plasma.

Amphibians have the same basic sources of water as most other animals: preformed water in food, preformed water taken in as “drink,” and metabolic water. For the most part, adult amphibians are carnivores. Their food is therefore succulent, but it yields a lot of urea, which they cannot excrete in concentrated form. Significantly, if an amphibian is eating insects, the total amount of water it gets from its food (preformed and metabolic) is likely to be no more than about 15% of the amount it needs just to excrete the urea it produces from its food. This calculation emphasizes the overwhelming importance of “drink” as a water source for the majority of amphibians.

Most amphibians do not in fact drink, but instead absorb water across their skin. This absorption does not necessarily require im- mersion in water. Many species can gain water at substantial rates merely by pressing their ventral skin against moist soil, moss, or other substrates.24 A region of the ventral skin at the posterior of the abdomen and extending onto the thighs—called the pelvic patch or seat patch—is often specialized for rapid water uptake. Its water permeability is modulated by insertion and retrieval of aquaporins in the cell membranes;25 when aquaporins are inserted, the water permeability of the patch cells is increased. A medium-sized and well-hydrated leopard frog (Rana pipiens) sitting on wet soil in its native habitat might well absorb 6–10 g of water per hour from the soil across its ventral surfaces, while it simultaneously loses a like amount into the air by evaporation across its dorsal surfaces—a dramatic display of dynamism!

When they are away from sources of water, most terrestrial amphibians are able to ward off dehydration for a time by using their bladder as a canteen. If an animal starts to dehydrate, the cells in the walls of its bladder are rendered permeable to water by aquaporin insertion in the cell membranes. NaCl is actively transported out of the bladder, thereby removing solute from the bladder contents and promoting osmotic outflux of water. The capacity of the bladder to hold fluid in terrestrial frogs and toads is remarkable: The water contained in the filled bladder is equal to 20%–50% of an animal’s bladder-empty weight. By contrast, in strictly aquatic amphibians, the bladder is usually tiny.

HOrMONAl CONTrOl OF rESPONSES TO dEHydrATiON In terrestrial amphibians, the neurohypophysial hormone arginine

24 Amphibians are far from being the only animals that take up water from moist substrates. The phenomenon has been documented in certain isopods, millipedes, insects, spiders, scorpions, land crabs, and snails.

25 Such aquaporins are regulated acutely in a cell by trafficking between intracellular locations where they are nonfunctional and the cell membrane where they enhance the permeability of the cell to transcellular osmosis.

Animals on land: Case Studies

Now let’s look at the water relations of some particular groups of animals on land. Doing so will provide an opportunity to integrate the points made in the previous section and discuss some new features of interest and importance. Chapter 30 continues this discussion of case studies with a focus on large-bodied mammals in deserts and dry savannas, such as camels and oryxes.

Amphibians occupy diverse habitats despite their meager physiological abilities to limit water losses

The terrestrial amphibians provide an instructive case study be- cause they have invaded an impressive variety of habitats, from the shores of ponds to, quite literally, deserts. Yet despite this diversity of habitats, most species are humidic animals that, regardless of where they live, are remarkably similar to one another in their physiological water-balance characteristics. Their diversification into a wide range

Water and Salt Physiology of Animals in Their Environments 771

vasotocin (see Table 16.2), called antidiuretic hormone (ADH), ac- tivates a suite of coordinated responses that collectively retard or reverse the process of dehydration. Release of ADH is stimulated if the volume of the body fluids (e.g., blood plasma) is decreased or if their osmotic pressure is increased. An amphibian’s overall reaction to ADH has appropriately been called the amphibian water-balance response. In its complete form (not shown by all spe- cies), this response involves changes at three sites in the body: the kidneys, bladder, and skin. First, ADH causes the kidneys to reduce their rate of urine production and elevate the urine concentration toward isosmoticity with the blood by mechanisms discussed in Chapter 29 (see Figure 29.5). Second, ADH stimulates the bladder cells to increase their rate of NaCl reabsorption and their perme- ability to water by aquaporin insertion in the cell membranes, responses that augment return of water from the bladder contents to the blood. Finally, ADH causes the ventral skin through which water absorption occurs to increase its capacity for water influx— facilitating rehydration—by stimulating increased blood flow and aquaporin insertion in the cell membranes.

ADH is by no means the only hormone active in water–salt physiology. For example, hydrins synergize with ADH in some contexts. Angiotensin II (see page 451) has been shown to be a principal controller of “cutaneous drinking” in frogs, stimulating the animals to press their ventral skin against moist substrates.

HOW dO dEHydrATiON-PrONE AMPHibiANS livE iN dESErTS?

As already mentioned, some species of frogs and toads, such as Bufo cognatus and the spadefoot Scaphiopus couchii in North America, have skin that provides no more protection against evaporative water loss than that of semiaquatic frogs such as leopard frogs (see Figure 28.19) yet live successfully in deserts or other arid habitats. Such desert species are in fact remarkably similar to the majority of terrestrial amphibians in all physiological respects, although some species show modest quantitative improvements over amphibians that live in moist habitats, such as by having a larger bladder, a somewhat greater tolerance of dehydration, or an accelerated pace of rehydration.

Behavior and seasonal dormancy are critical keys to the suc- cess of these desert amphibians. Dehydration can kill them in an hour—or just a few hours—if they are exposed in the desert. Stringent behavioral control of water loss is therefore a requirement of life. These desert amphibians spend much of their time in protective microhabitats, especially in burrows underground, and are largely nocturnal. They also employ seasonal dormancy to simply “retire from the scene” and protect their water status during dry seasons. Spadefoot toads (S. couchii), for example, spend many months of each year in dormancy. Overall, these desert amphibians are reclusive animals, holed up in secluded places during much of their lives. For some, dormancy dominates their lives more than activity. Their reward is that they are able to survive in deserts despite the high permeability of their skin and other vulnerabilities.

“rAdiCAl” PHySiOlOgiCAl SPECiAlizATiONS OCCur iN SOME ArbOrEAl, Arid-zONE FrOgS For an amphibian to exist in arid places without being restricted to a secluded life, it must have evolved superior physiological mechanisms to cope with the challenges of dehydration stress. Biologists are gradually learning more about such mechanisms in several types of unusual

FigurE 28.22 Arboreal frogs of the genus Phyllomedusa spread protective lipids secreted by integumentary glands over their skin surface The lipids sharply reduce the rate of evaporative water loss across the frogs’skin.The spreading is carried out by a series of stereotyped limb movements, as shown. (After Blay- lock et al. 1976.)

arboreal frogs that live exposed lives in arid or semiarid habitats. Frogs of this sort in two genera—Phyllomedusa of South and Cen- tral America and Chiromantis of Africa—have been studied for several decades and are known to have physiological abilities to conserve water that are extraordinarily different from those of most amphibians. One distinctive trait of these frogs is that their integu- mentary permeability to water is exceptionally low; their rates of evaporative water loss are consequently little different from those

of some lizards of similar size.26 Cutaneous lipids are responsible

Hill Animal Physiology 4E
for this low skin permeability in both genera. The lipids are spread

Sinauer Associates
Foignutrehe28o.2u2tsi1d2e-2o3f-1th5e skin rather than being incorporated within

the skin tissue. Phyllomedusa, for example, secretes lipids (mainly waxy esters) from skin glands and spreads them on its skin surface (FigurE 28.22). A second highly distinctive trait of these genera is that they excrete much of their nitrogenous waste as poorly soluble uric acid or urates (80% in P. sauvagei, for example).

The tiny, dramatically colorful reed frogs (Hyperolius) of the African savannas represent another remarkable group of arboreal frogs—a group that is only now starting to be well understood. Some species have exceptionally low skin permeabilities. They do not, however, routinely produce uric acid or other related compounds as do the frogs just discussed. During the dry season, reed frogs remain in exposed locations on the branches of bushes and trees even as they undergo profound dehydration. They stop producing

26 Expressed in the same units as used in Table 28.6, the resistance to evaporative water loss of the skin in species of Phyllomedusa and Chiromantis is generally 200–400 s/cm.

772 Chapter 28

urine at such times, and much of their waste nitrogen accumulates in their body fluids as urea. However, as they dehydrate, they start to synthesize guanine from the waste nitrogen retained in their bodies; in H. viridiflavus, 25% of waste nitrogen becomes guanine. Guanine is a low-solubility purine like uric acid (see Figure 29.24). Remarkably, the frogs deposit much of the guanine they synthesize in skin cells (iridophores), and the little animals turn bright white from its presence. The formation of guanine lengthens the time the frogs can store waste nitrogen (because it keeps the nitrogen out of solution), and it reduces solar heating by increasing the reflectance of the skin to incoming radiation!

xeric invertebrates: because of exquisite water conservation, some insects and arachnids have only small water needs

Certain insects are among the most successful of all animals in severe desert conditions. Being successful, they provide succulent food for other, less-adept desert dwellers.

Certainly one of the most intriguing phenomena in the living world is presented by desert ants that feed on other desert insects killed by heat. Life for diurnal insects in severe deserts is so tenuous that, every day, some individuals accidentally die of overheating despite extraordinary adaptations for desert existence. Desert ants of several species scavenge the bodies of such heat-killed insects. The deaths often occur in the heat of the day. Thus, to get moisture from their prey, the ants must venture forth from benign underground burrows in the heat of the day to gather the dead bodies before the sun quickly bakes the bodies dry. Species of such ants in deserts around the world have independently evolved unusually long legs (FigurE 28.23). This is believed to be related to the fact than air temperature declines extremely steeply with altitude above the sun-heated sand. The stiltlike legs—although they elevate an ant’s body just millimeters higher than it would otherwise be—can reduce the air temperature to which the body is exposed by 10°C. The ants also exploit the steep temperature gradient at times by taking breaks from desert foraging to climb up on pebbles or other high points in the desert terrain to reach even lower air temperatures than their stiltlike legs permit. These ants can tolerate tissue temperatures of 52°C–55°C (126°F–131°F). They

FigurE 28.23 diurnal desert ants that collect heat-killed in- sects are noted for long, stiltlike legs that keep them above the intensely hot sand surface Shown is Cataglyphis diehli. (Photograph by Rüdiger Wehner.)

thus rank with the most heat-tolerant of all animals. Nonetheless, they heat up promptly under intense sun and can easily suffer heat death themselves if they fail to act quickly as they exit their burrows, seek out recently heat-killed prey, and return to underground safety. Accurate navigation is crucial: After ants have traveled hundreds of meters to find prey in featureless deserts, they must find their burrows again. Ants of the genus Cataglyphis in the Sahara Desert have been shown to have evolved one of the most remarkable of animal navigation systems despite having a brain that weighs 1/10 of a milligram (see Figures 18.3 and 18.4).

Deserts are just the driest of the habitats in which insects and arachnids (e.g., spiders and scorpions) live. Some species prosper in a variety of other arid places. The suite of characteristics that permits many species to prosper in semiarid and arid habitats includes several physiological attributes that promote highly effective water conserva- tion: high integumentary resistance to water loss (provided chiefly by epicuticular lipids); stringent limitation of respiratory water loss by control of the opening of the spiracles; excretion of waste nitrogen in poorly soluble forms; and an ability (at least in many insects) to produce concentrated urine (maximum osmotic U/P = 2–8).

Some flightless insects, ticks, and mites, in addition, have a way to obtain water that is unique in the animal kingdom: They are able to gain water from the gaseous water vapor in the air in a steady man- ner while they are at the same temperature as the air. For example, the desert cockroach Arenivaga investigata can gain water from the air steadily even when the ambient relative humidity is as low as 79%–83%. The mealworms (Tenebrio) that people often use as food for pets can gain water down to 88% relative humidity, and firebrats (Thermobia) can do so down to 45%. A water gain of about 10% of body weight per day is the rule when these insects are dehydrated and living in humid air. The mechanism of water uptake in many cases remains subject to debate. The site of uptake in the desert cockroach and some ticks is the mouth. Mealworms and firebrats, by contrast, absorb water via the rectum. Some investigators believe that true, primary active transport of H2O sometimes occurs in these arthropods, although most disagree. A mechanism that is known to operate in some species is the production—at the mouth or rectum— of localized, superficial pockets of body fluids with such high solute concentrations that they have water vapor pressures below ambient water vapor pressure;27 water vapor diffuses from the atmosphere into such body fluids, following the vapor pressure gradient.

Few water budgets have been worked out for insects or arach- nids, but it is clear that certain species are so effective in limiting water losses that they can maintain water balance at moderate temperatures and low humidities while having no drinking water and eating only air-dried foods. Common examples include grain beetles and clothes moths. Besides the small amounts of preformed water in air-dried foods, the only sources of water for such insects are metabolic water and whatever water they may gain from atmospheric water vapor.

Because of their short generation times, insects are among the most useful animals for studies of physiological evolution using laboratory populations. Experiments using insects demonstrate that the resistance of animals to water stress can respond dramatically to evolutionary selective pressures (bOx 28.6).

27 Recall from Chapter 5 that the water vapor pressure of a solution is a colligative property and decreases as solute concentration increases.

water. Many species of small mammals in deserts also acquire sub-

stantial quantities of preformed water in their foSoindaubercAasussoeciathteesy
Box Figure 28.06 12-23-15

eat primarily insects or plants; in North America, these animals include pack rats, grasshopper mice, and ground squirrels. In all the major deserts of the world, however, there have evolved extraordi- nary species of small mammals that live away from drinking water and eat principally air-dried seeds or other air-dried plant matter. The kangaroo rats (Dipodomys) of North America’s southwestern

28 See Chapter 30 for a discussion of camels, oryxes, and other large mammals in deserts and dry savannas.

Water and Salt Physiology of Animals in Their Environments 773

xeric vertebrates: Studies of lizards and small mammals help clarify the complexities of desert existence

When one treks to the driest deserts, one sees only a few sorts of

animals leading active lives. Lizards and small mammals28 are

two groups that especially stand out, in addition to insects and

arachnids. A fact that draws interest to both the lizards and small

mammals is that they often have no chance of finding drinking

water for months on end. Because they cannot travel far, they have

drinking water only when local rains provide it, and rains come only

sporadically. Desert lizards characteristically eat insects or living

plant tissues, foods that supply significant amounts of preformed

deserts (see the opening photo of Chapter 29) provide classic ex- amples. Others include the kangaroo mice and pocket mice of North America, some gerbils and jerboas of Old World deserts, and the hopping mice of Australian deserts.

A significant, intriguing trait of the lizards that prosper away from drinking water is that, like other lizards, they are diurnal. The key traits that permit the existence of lizards as diurnal animals in the driest places on Earth include, first, their relatively low metabolic rates (see Figure 7.9). A low metabolic rate not only can greatly reduce water losses, it also reduces food needs—an asset in habitats where populations of food organisms are themselves stressed and relatively unproductive. Second, the desert lizards excrete their nitrogenous wastes as water-sparing uric acid or urates. They also

Hill Animal Physiology 4E

use behavior to avoid stresses that are avoidable, as by moving into underground burrows or shadows during the heat of the day. Some species employ salt glands. Finally, desert lizards tend to be remarkably tolerant of large shifts in their body-fluid composition, such as high blood solute concentrations, during dehydration. They can survive perturbations of their body fluids—often for long periods—that would kill a mammal or bird.

The small mammals of deserts operate on a different, higher scale of metabolic intensity than the lizards. High metabolic intensity is in

774 Chapter 28

itself a liability in deserts; it raises the rate of respiratory evaporative water loss, as we have seen, and it can contribute enough endogenously produced heat to add significantly to heat stress. Small mammals that live in deserts have, in general, evolved lower basal metabolic rates than nondesert mammals of the same body size. Some species, moreover, undergo daily torpor or estivation when they are short of food or dehydrated. Still, when small mammals are active, their metabolic rates are far higher than those of lizards.

The species of small desert mammals that, by far, have attracted

the most curiosity are the ones that eat primarily air-dried plant mat-

ter. In classic studies 60 years ago that contributed to the genesis of

modern animal physiology, researchers produced the water-balance

summary for kangaroo rats (D. merriami) in FigurE 28.24. As we

discuss this information, it will be important to keep in mind that the

animals were studied at 25°C (77°F). They had no drinking water and

were fed only barley grain. Each of the five lines in the figure shows

how a key attribute of their water physiology varied with the humidity

of the air. The red lines show the animals’ minimum water losses by

evaporation and elimination of urine and feces. Kangaroo rats have

several specializations for conserving water. They exhibit exceptionally

low cutaneous permeability to water; they cool their exhalant air by

nasal countercurrent exchange; they can produce very concentrated

urine (osmotic U/P = 14); and they can restrict their fecal water losses

exceptionally. Their minimum evaporative, urinary, and fecal water

losses are stacked on top of each other in the figure so that the heavy red

line at the top represents their total water losses. Evaporative losses

decrease with increasing humidity, but minimum urinary and fecal

losses are independent of humidity, so the animals’ total water losses

decrease as humidity increases. The blue lines show the water inputs

of the kangaroo rats. Their production of metabolic water is the same

at all humidities because it depends on metabolic rate, which is the

same regardless of humidity. In contrast, the rats’ input of preformed

water increases as humidity increases because air-dried plant matter,

such as barley grain, comes to equilibrium with the water vapor in the

air and contains more water when the humidity is high. The animals’

inputs of metabolic and preformed water are stacked so that the heavy

blue line represents their total water intake.

Hill Animal Physiology 4E Kangaroo rats can be in water balance if their total water inputs

28.24, therefore, the animals can be in water balance while eating air-dried grain and drinking nothing if the relative humidity is above about 10%. Most of their water input is metabolic water. As stressed in Chapter 27 (see page 736), this is not because they produce exceptional amounts of metabolic water. It is because they conserve water so well that metabolic water can meet most of their needs.

Now we need to recall that the studies in Figure 28.24 were done at 25°C. The researchers who carried out the studies obtained information on soil temperatures and nocturnal air temperatures in the parts of the southwestern deserts of the United States where they worked.29 They concluded that kangaroo rats in those deserts are usually not exposed to temperatures higher than 25°C. Being nocturnal, the kangaroo rats live in their cool burrows during the heat of the day and emerge onto the desert surface only in the cool of the night. Thus the researchers concluded—based on Figure 28.24—that kangaroo rats are able to stay in water balance in the desert while eating air-dried seeds or other air-dried plant material and drinking nothing.

29 For soil temperatures they used the information in Figure 1.18.

Kangaroo rats can stay in water balance if the humidity exceeds that at which the “total water intake” line crosses the “total water loss” line.

equal or exceed their total, minimum water losses. Based on Figure

Sinauer Associates

0 20 40 60 80 100 Relative humidity (percent) at 25°C

FigurE 28.24 A kangaroo rat water budget For this study, carried out at 25°C, kangaroo rats (Dipodomys merriami) were fed husked barley grain at equilibrium with atmospheric moisture and provided no drinking water (they usually do not drink even if water is available).The water losses shown (red lines) are the minimum pos- sible water losses; in actuality, if water intake exceeds minimum losses, the animals increase their losses (as by excreting more-dilute urine) so that losses match inputs (blue lines).The amounts of water graphed on the y axis are those gained or lost each time 100 g (dry weight)

of barley is consumed. Under the conditions of study, the animals normally consume 100 g of barley in about a month. (After Schmidt- Nielson and Schmidt-Nielsen 1951.)

How is the water physiology of kangaroo rats affected if the ambient temperature is different from 25°C? The dominant modes of water gain and loss are metabolic water production (MWP) and evaporative water loss (EWL) (see Figure 28.24). A straightforward (although not complete) way to gain insight into the effect of tem- perature on water balance is to examine how MWP and EWL relate to each other as temperature varies. The two are often expressed as a ratio of gain over loss—MWP/EWl—for this purpose. The numerator, MWP, tends to increase as ambient temperature de- creases because MWP varies with metabolic rate, which increases as temperature decreases below the thermoneutral zone (see Figure 10.28). The denominator, EWL, by contrast, typically tends to decrease as ambient temperature decreases in small mammals and birds. The MWP/EWL ratio (water gain over water loss) therefore becomes dramatically more favorable as the ambient temperature falls (FigurE 28.25).

Based on this analysis of the MWP/EWL ratio, we would expect kangaroo rats in the wild to be under far less water stress during the cool seasons of the year than during the warm seasons. In fact, studies of kangaroo rats (D. merriami) in the wild match this expectation. In one population, the urinary osmotic pressure of the rats averaged about 1000 mOsm (U/P = 3) in midwinter but rose to about 4000 mOsm (U/P = 11) in midsummer. Low winter temperatures apparently placed the rats in such a favorable situa-

Figure 28.24 12-23-15

70

60

50

40

30

20

10

Gain from preformed water in food

Loss to feces Loss to urine

Loss to evaporation

00 5 10 15 20 Absolute humidity (mg H2O/L air)

Grams of water per 100 g of dry barley metabolized

3

2

1

0

FigurE 28.25 An index of water balance: metabolic water production (MWP) as a ratio of total evaporative water loss (EWl) This ratio provides a useful (although incomplete) index of ability to stay in water balance for species in which metabolic water production and evaporation are the principal processes of water gain and loss. Shown are data for two populations of kangaroo rats, Dipodomys merriami, and two species of desert birds, the dune lark (Mirafra erythrochlamys) and zebra finch (Poephilia guttata). (Data from Tracy and Walsberg 2001; Williams 2001.)

tion that they had a water surplus and didn’t need to concentrate their urine maximally.

Although most populations of kangaroo rats live in places where they experience the conditions we have discussed up to now, some populations live in far hotter places (in the Sonoran Desert) where the animals probably encounter temperatures as high as 35°C (95°F) both in their burrows and on the desert surface at night. These animals cannot achieve water balance while eating air-dried foods, and they can’t make up the difference by drinking because no drinking water is present. Fortunately insects are able to exist and accumulate body fluids in this severe environment. The kangaroo rats probably achieve water balance by adding insects and green- plant parts to their usual diet of air-dried seeds: a striking example of one species taking advantage of adaptations of other species.

FigurE 28.26 lark species along a gradient of wa- ter availability Water availability in a species’ habitat is quantified by an index that can vary from 1.5 (very arid) to 3.5 (moist). In each graph, each symbol represents a different species of lark. Metabolic rates and rates of water loss are expressed using indices designed to be indepen- dent of body weight. Metabolic rates shown are basal metabolic rates (red) and average free-living metabolic rates (black). Rates of water loss shown are rates of total evaporative water loss (red) and free-living water turn- over rates (black). Activity is percentage of daylight hours spent active. Shown in the photograph is a desert-dwell- ing hoopoe lark (Alaemon alaudipes), one of the species found in the most arid habitats. (After Tieleman 2005.)

0 10 20 30 40 Ambient temperature (°C)

Water and Salt Physiology of Animals in Their Environments 775 xeric vertebrates: Some desert birds have

specialized physiological properties

The fundamental conflict between heat balance and water balance in hot deserts is emphasized by the study of birds because most desert birds are diurnal and therefore do not evade the heat of the day. Despite the fact that birds often confront the stresses of deserts head-on, the species of desert birds first studied seemed to exhibit remarkably few specializations for desert existence. Birds as a group have higher body temperatures than mammals, are especially tolerant of hyperthermia, synthesize uric acid, and can fly to distant water- ing places. Such traits, shared by birds as a group, seemed initially to be sufficient for successful desert existence. A dogma developed that the presence of birds in deserts is largely a consequence of a happy marriage between the standard features of all birds and the requirements of desert life.

Physiologists have recently come to realize that this dogma is not correct. For example, when data on large numbers of species are analyzed statistically, desert birds turn out, on average, to be systemi- cally different from other birds. One difference is that desert birds tend to exhibit relatively high resistance to evaporative water loss.

A particularly instructive comparative study is being carried out on a group of closely related birds, the Old World larks, which occupy an extreme diversity of habitats. Whereas certain species of Old World larks occur in moist habitats, others occur in semiarid places, and some live in hot, hyperarid deserts. The hoopoe lark (Alaemon alaudipes), seen in the inset in FigurE 28.26, exempli- fies the latter. When hoopoe larks are living in hyperarid deserts such as those of the Arabian Peninsula, they eat insects and other arthropods and thereby get preformed water from their food, but

Lark species native to arid habitats exhibit particularly low rates of metabolism and water turnover, compared with lark species native to moist habitats. Moreover...

...arid-habitat larks are also less active and have fewer clutches of young than moist-habitat larks.

100 80 60 40 20 00 4

5 4 3 2 1

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3 2 1

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2.5

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EWL

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MWP/EWL

776 Chapter 28

they never drink—water is almost never within flight distance. As Figure 28.26 illustrates, when diverse species of larks are arrayed along an axis of water availability—with a water-availability index of 1.5 representing a very arid habitat and an index of 3.5 repre- senting a moist habitat—the species native to dry habitats display distinctive features. Included are physiological specializations: They exhibit low metabolic heat production and low water turnover. The low water turnover of the dry-habitat species is accounted for, in part, by cutaneous lipids that, in comparison with those of other larks, are unusually protective, and this difference in water loss between desert birds and other birds correlates with differences in the composition of their cutaneous lipids.

These new revelations in the study of birds help emphasize that although molecular biology is one of today’s most important cutting edges, the comparative study of related species (the products of evolution) in divergent habitats remains a powerful source of insight for understanding life on Earth.

sometimes a principal player, as in the control of avian salt glands. Some of the most important hormones involved are the antidiuretic and diuretic hormones. diuresis is the production of an abundant (and usually dilute) urine. A diuretic hormone promotes diuresis. An antidiuretic hormone opposes diuresis or, in other words, modulates the excretory organs so that a relatively low volume of (usually concentrated) urine is produced.

Vertebrates are considered to produce only antidiuretic hor- mones. In insects, however, diuretic as well as antidiuretic hormones occur. Certain blood-sucking insects, for example, have a diuretic hormone that is secreted after a blood meal, promoting rapid excre- tion of much of the water in the blood and thereby concentrating the nutritious part of the meal (e.g., proteins) in the gut.

In vertebrates, which will be our focus in this brief discussion, three hormones or types of hormones play particularly important roles in the regulation of water–salt physiology:

1. Antidiuretic hormone (AdH), produced by the neurohypophysis (see pages 449–451 and Table 16.2)

2. Mineralocorticoids, most notably aldosterone, produced by the adrenal cortex or homologous interrenal tissue (see page 440)

3. Natriuretic hormones (see page 451)

Focusing on ADH first, its principal effect in mammals—and its principal effect on the kidneys in other terrestrial vertebrates—is to control the renal excretion of pure water (osmotically free water) relatively independently of solute excretion. To see this point, let’s use a mam- mal as an example. Consider that a mammal has a certain quantity of urea, salts, and other solutes that it must excrete per day. If the solutes collectively are excreted at the maximum concentration the individual can achieve, the accompanying water loss can be considered to be strictly obligated by solute excretion. However, if the solutes are excreted at less than maximum concentration, then the urine contains additional water that is not obligated by solute excretion; in essence, the urine is diluted by the addition of pure water above and beyond the amount needed to void solutes, and the additional water represents a specific excretion of water itself. The urine can therefore be considered to consist of two components: (1) the solutes and their associated water and (2) a quantity of additional pure water. ADH controls the magnitude of the latter component. If a person, for instance, has a constant daily solute output, but consumes little water on one day and a lot of water the next day, ADH is secreted on the first day, and—because of its antidiuretic effect—restricts the amount of water excreted with the solutes. However, ADH secretion is reduced on the second day; this permits diuresis and thus the excretion of a great deal of water with the solutes.30

The principal effect of aldosterone is to cause the kidneys to hold back Na+ from excretion while promoting the excretion of K+ in the urine. These actions of aldosterone most obviously affect the quantities of Na+ and K+ in the body fluids. Less obviously, the action of aldosterone is one of the most important factors in the routine regulation of extracellular-fluid volume (including blood volume). To understand this latter role of aldosterone, consider that Na+ is largely excluded from intracellular fluids by being actively transported out of cells (see Figure 5.10). Increases or decreases in the amount of

30 The action of ADH is discussed in more detail in Chapters 16 and 29.

Control of Water and Salt balance in

Terrestrial Animals

The kidneys and other organs responsible for water and salt regula- tion in terrestrial animals are generally under hormonal control in both vertebrates and invertebrates, although the nervous system is

Na+ in the body therefore lead to quantitatively similar increases and decreases in the amount of Na+ (and accompanying anions, notably Cl–) in the extracellular fluids. When Na+ is retained in the body by being held back from the urine—and Na+ thus accumulates in the extracellular fluids—the systems controlling the osmotic pressure of the extracellular fluids cause water to be retained as well, so as to maintain a normal extracellular-fluid osmotic pressure. In that way, the extracellular-fluid volume is expanded. Conversely, if the body’s Na+ content is reduced, water from the extracellular fluids is excreted to maintain a stable extracellular-fluid osmotic pressure. Actually, although aldosterone has its major effects on the kidneys, it often functions as an all-purpose Na+-retention hormone. In mammals, for example, it stimulates the salivary glands, sweat glands, and intestines to increase reabsorption of Na+. Moreover, aldosterone stimulates salt appetite.

The natriuretic hormones—often termed natriuretic peptides because they are types of peptides—promote addition of Na+ to the urine, as their name indicates (natri, “sodium”; uretic, “having to do with excretion in the urine”). Despite an explosion of knowl- edge since 1990, many uncertainties remain about the functioning of the natriuretic hormones because there are multiple chemical forms—each of which potentially has multiple effects—which can differ from one set of vertebrates to another. The heart and certain brain regions (e.g., hypothalamus) are the chief sites of secretion in mammals. The atrial natriuretic peptide (ANP) of mammals is the best understood of these hormones. It is called atrial natriuretic peptide because it is produced principally by the atria of the heart (see page 451). Mammalian ANP has actions that in many ways are opposite to those of aldosterone. It inhibits aldosterone secretion and directly modulates the kidneys to promote Na+ excretion by increasing both urine volume and urine Na+ concentration.

The mechanisms of control of the secretion of ADH, aldoste- rone, and ANP are complex and incompletely understood. All of these hormones participate in negative feedback systems—now discussed—that act to stabilize the volume and osmotic pressure of the extracellular body fluids.

For the volume of the extracellular body fluids to be regulated, either the volume itself or reliable correlates of volume must be sensed, so that the regulatory systems will “know” whether to promote an increase or a decrease in volume at any particular moment. Prob- ably volume itself is not sensed. However, good evidence exists that certain correlates of volume are sensed; for example, both the blood pressure and the extent to which blood-vessel walls are stretched are functions of blood volume, and pressure and stretch receptors that participate in volume regulation are known (e.g., in and around the heart). Similarly, if the osmotic pressure of the extracellular body fluids is to be regulated, either it or close correlates must be sensed. No doubt exists that receptors for osmoregulation are present (e.g., in the hypothalamus), but whether they respond to osmotic pressure itself, Na+ concentration, or other correlated properties remains debated.

Secretion of ADH is controlled in part by changes in blood volume; pressure sensors and other sensors of volume affect ADH secretion by way of nervous inputs to the hypothalamus and also via the renin–angiotensin–aldosterone system (see next paragraph). Decreases in blood pressure activate ADH secretion, a response favoring fluid retention. Secretion of ADH is also under the control of osmoreceptors or other detectors of the concentration of the body fluids. Increases in the osmotic pressure of the body fluids

induce increased ADH secretion; the ADH then favors the specific retention of water by the renal tubules, thereby tending to lower the osmotic pressure of the body fluids.

Aldosterone secretion is controlled to a major extent by another hormonal system, the renin–angiotensin system (see Figure 16.17), which itself is partly under the control of blood-pressure receptors and other detectors of blood volume. A decrease in blood pressure, signifying a reduction in blood volume, activates secretion by the kidneys of the hormone renin (pronounced “ree-nin”), which in turn causes formation in the blood of angiotensin II. The angiotensin stimulates the adrenal glands to secrete aldosterone, which induces increased Na+ reabsorption from the urine, tending to expand extracellular-fluid volume and raise blood pressure.

Secretion of ANP is stimulated by expansion of extracellular- fluid volume, which is detected by stretching of the walls of the atria in the heart. ANP then promotes loss of extracellular fluid. One of ANP’s principal overall effects is a decrease in blood pressure.

Water and Salt Physiology of Animals in Their Environments 777