Gnathostomata. The blood is isosmotic to sea How to regulate in sea water

4.4.3 Gnathostomata. The blood is isosmotic to sea How to regulate in sea water

water and concentrations of sodium and chloride Sea water has an osmolality of about 1000 mosmol. ions are the same as in sea water, but are slightly Among marine fish three different strategies for higher in the tissues due to high levels of intracel- regulation of internal water and total solute con- lular amino acids. Osmotic water exchange is centrations can be distinguished:

more or less absent, while water exchange rates

1 same osmolality as sea water, no regulation at may be as high as 2.3 l kg -1 h -1 (McInerney 1974) all (hagfish);

due to the free permeability to water. Urine pro-

Physiology of Living in Water

duction is minimal, and chloride cells have been produce urine more concentrated than the blood so observed. Though hagfish are strictly marine that excess salts are removed and water is con- (stenohaline), experiments by McInerney (1974) served. In fact many marine teleosts have evolved demonstrated that they have the ability to reab- an aglomerular kidney to minimize water loss, and sorb sodium from the glomerular filtrate, a feature urine flow rate is as low as 1–2% of body weight per necessary for living in fresh water. This could be day. Marine fish have solved the osmotic problem reminiscent of an original freshwater ancestry as follows (Potts 1976; Kirsch et al. 1981). After (Marshall and Smith 1930).

drinking, the water passes into the oesophagus, which is impermeable to water but permeable to light monovalent ions. Sodium and chloride thus

diffuse into the blood down their concentration Like the hagfish the elasmobranchs are not in gradients. When entering the intestine, which is danger of losing water because they are isosmotic permeable to water, the water has become hypos- to the medium. However, like the marine teleosts motic to sea water and nearly isosmotic to the they are hypo-ionic to sea water. Plasma os- blood. A Na + /K + /2Cl - cotransport mechanism molarity is increased to seawater levels by the located in the apical brush border membrane trans- organic osmolytes, urea and TMAO. Urea is pro- ports the ions from the gut lumen into absorptive duced from the nitrogen waste product, ammonia, cells and water follows passively in a process called in the ornithine–urea cycle in the liver, from where solute-linked water transport. Na + /K + ATPase, it enters the plasma and other body fluids. Though which is present in the basal membrane of the urea is less toxic than ammonia, the levels seen in absorptive cells, provides the energy for this active elasmobranch plasma would be fatal for other fish transport. The excess sodium, potassium and chlo- except Latimeria. Elasmobranchs have achieved ride ions are then transported in the blood to the higher tolerance to urea by retaining TMAO, gills where they are excreted via the ‘chloride cells’ which counteracts the effect of urea at a ratio of (Fig. 4.4). The concentration of divalent ions is low about 1 : 2 in their body fluids, and by having en- in sea water; 80% of these ions that are taken up by zymes and proteins that appear to be less sensitive drinking are expelled in the faeces. The remaining to disruption by urea. A special sodium chloride ions are excreted in the urine. excretory organ has evolved in the elasmobranchs.

Marine elasmobranchs

In order to become truly marine the teleosts had This so-called rectal gland uses a Na + /K + /2Cl - co- to solve the osmotic problems for the spawned transport mechanism to secrete excess sodium egg. At spawning, marine fish eggs must contain a and chloride ion intake from ingested food. The water reservoir to compensate for the passive secretion contains very little of the organic water loss imposed by the hyperosmotic sea water osmolytes and is isosmotic to the plasma. Energy (Fyhn et al. 1999). The high water content of the for the cotransporter is provided by an abundance yolk of marine teleost eggs forms this water of Na + /K + ATPase in the gland.

reservoir. Regardless of systematic affinities, most extant marine fishes spawn pelagic eggs. Yolk protein hydrolysis and increase in content of free

amino acids during final oocyte maturation is part In contrast to marine elasmobranchs the marine of the mechanism that brings water into the yolk teleosts, being hyposmotic to sea water, lose water before the eggs are spawned (Rønnestad et al. 1996; to the medium primarily over the thin gill epi- Thorsen & Fyhn 1996). thelium. They have to replace this water loss and drink between 10–20% and 35–40% of their body weight per day. In addition there is a constant pas-

Marine teleosts

4.4.4 Antifreeze

sive influx of monovalent ions over the gills as well For hagfish, marine elasmobranchs and freshwater as dietary salt uptake. The kidneys are not able to teleosts, freezing is not a problem since they have

Secondary lamellae

Negative charge

3 mM K ~ – 140 mM Cl

Na + K +

Sea water

Na +

K – + 2Cl

470 mM Na ~ + Blood

10 mM K ~ + channels

~ – 560 mM Cl

Positive

Site of

α-chloride cell:

charge

Na + -K + -ATPase

in fresh and sea water Apical pit

Fig. 4.4 Schematic illustration of Pillar

part of secondary lamellae on a gill cells

Tight junction

Leaky junction

filament showing the two types of chloride cells involved in the active secretion of chloride ions.

Accessory

The process depends on Na + /K +

cell

Gill filament

ATPase activity and causes

β -chloride cell:

polarization of the cell. Sodium

in fresh water

ions may diffuse out via the leaky junctions.

body fluids that are either isosmotic or hyperos- motic to the surrounding water, as long as the

4.5 RESPIRATION AND

water is not frozen. Marine teleosts, however, are

SPECIAL ADAPTATIONS FOR

hyposmotic to the ambient water and can freeze to

LIVING IN LOW OXYGEN

death when the water temperature drops below

4.5.1 The environment

0 °C. To prevent freezing the fish may produce

macromolecular ‘antifreeze’ compounds in their Fish live in a dense and very viscous environment, blood serum. The antifreeze consists of glycopro- which for a terrestrial animal would appear to be teins or proteins, which coat and isolate ice crys- almost depleted of oxygen. At best, one volume of tals forming in the blood by binding their hydroxyl water contains a little less than 4% the amount of groups to oxygen molecules on the surface of the oxygen present in the same volume of air. In air ice crystals (DeVries and Wohlschlag 1969). The the solubility of a gas would be roughly 1000 ml/ production of antifreeze is dependent on cold accli- 101.3 kPa = 9.87 ml l -1 kPa -1 (at 15 °C). In practice mation and short photoperiods (Duman and De- the respiratory gases are treated as ideal gases, Vries 1974a), and genetically based differences which means that oxygen and carbon dioxide in antifreeze production have been described would dissolve more or less equally in air. In water, (Duman and DeVries 1974b). In the aglomerular however, the solubility of gases depends on: (i) the kidneys of Antarctic fish the glycoproteins are nature of the gas, (ii) the pressure of the gas in the conserved rather than filtered out of the blood, gas phase, (iii) the temperature and (iv) the pres- which lowers the energetic cost of osmoregulation ence of other solutes. Thus, while the solubility of in these fish (Dobbs et al. 1974; see also Section oxygen in water is only about one-thirtieth the sol- 4.5.4).

ubility in air, the solubility of carbon dioxide is

Physiology of Living in Water

more or less the same in air and water depending on

4.5.2 Gas exchange over gills

temperature and is similar at about 15 °C. For calculating gas fluxes we use the concept of All fish have a unidirectional flow of water over Ohm’s law: flux = capacitance ¥ potential differ- their gills, except for adult lampreys, which have ence, which gives the following three fundamental tidal ventilation in and out of the gill sac via the gas equations:

valved branchial openings. The mouth cannot be open since the lamprey is likely to be attached to

M ˙ CO 2 = V ˙ m [ b m ◊ ( P e - P i ) ] CO 2 (4.2) either rocks or hosts. Synchronous expansion and

contraction of the buccal opercular cavities venti- M ˙ O 2 = V ˙ m [ b m ◊ ( P i - P e ) ] O 2 (4.3) late the gills. This provides a nearly constant flow

of water over the gill surfaces. At a given swim- . where M gas is the volume of gas taken up (O 2 ) or . ming speed fish adopt ram ventilation to conserve

produced/released (CO 2 ), V m the flow of medium energy. The branchial pump is switched off and the over the gills, b m the capacitance coefficient (solu- flow of water over the gills is regulated by the open- bility) of the gas in the medium and P the partial ing of the mouth in relation to the actual speed and pressure of gas (i, before gills; e, after gills). From oxygen demand. The gills consist of bony or carti- these expressions, we may calculate:

laginous arches to which one row of paired gill filaments are anchored. Numerous secondary

M ˙ CO lamellae protrude from both sides of each fila-

= RQ (4.4) ment. A layer of thin epithelial cells covers the O 2 outside of the lamellae. Beneath the basement

membrane are supportive pillar cells and blood where RQ is the respiratory quotient (R, the ex- vessels running in the opposite direction to the

change ratio, is used when examining isolated water flow (Fig. 4.4). This arrangement ensures parts of the fish). Rearranging equations 4.2–4.4 very efficient gas exchange, with oxygen utiliza- and ignoring P i :

tion as high as 80% and postbranchial oxygen par- tial pressures higher than in the water passing

() b

() through the gill. The functional area of the gills can

e CO 2 2 = ◊ ( P e - P iO ) () b m CO

mO

2 be changed by shunts in the secondary lamellae or by the action of an autoregulatory system, which

If the fish extracts all the oxygen in the water (P i @ reacts on blood pressure by increasing the tonus

21 kPa, P e = 0 kPa), the partial pressure of carbon of the contractile elements in the pillar cells so dioxide can, at a maximum, be about 0.7 kPa in the re-routing blood flow (Laurent 1984). Intrinsic exhalant water. This means the maximum partial muscles in the gill filaments of most teleosts can pressure of carbon dioxide in the arterial or post- change the angle of the filaments on each arch and branchial blood will not exceed this value, which thus alter the flow pattern over the secondary is almost a factor of 10 lower than for air-breathing lamellae. animals.

This low oxygen availability has undoubtedly

Other sites for gas exchange

contributed to the evolutionary development of:

1 large gill surface areas for extremely efficient gas An efficient gas exchange system should have exchange;

the following characteristics: close contact to the

2 air-breathing organs and the necessary circula- medium, short diffusion distances, and a well- tory arrangements;

developed circulation that can transport oxygen

3 acid–base regulation depending more on ion efficiently from the site of gas exchange to the tis- than ventilatory exchanges.

sues. Cutaneous respiration may be of some signif- icance in a few cases either when oxygen demand

84 Chapter 4

is very low, as in some Antarctic fishes, or when or other changes. Aneural control is mainly ef- the diffusion distance is very short, as for fish fected by: larvae. In adult fish, cutaneous respiration is

1 changes in blood volume and venous return by less than 30% of routine metabolism (O. Brix, swimming movements and mobilization of blood unpublished results). Its significance is still poorly into general circulation from the spleen, liver or investigated, but it may be of some importance for blood sinuses in various species; fish like eels when migrating short distances

2 direct response of heart muscle (pacemaker) to across land (Berg and Steen 1965). For air-breathing temperature changes, which increase the heart organs see Section Air breathing.

rate (Randal 1970);

3 circulating catecholamines like adrenaline,

4.5.3 which stimulates heart rate and stroke volume Circulation and gas transport

and dilates the gill vasculature (Nakano and The main tasks of the circulatory system are to Tomlinson 1967; Bennion 1968), and noradrena- transport respiratory gases, nutrients and metabol- line, which constricts systemic vascular beds ic waste products, endocrine factors and heat. (Wood and Shelton 1975). Most fish have a single circulatory system. The

The hearts of teleosts and elasmobranchs, but heart consists of the sinus venosus, atrium, ven- not hagfish and lungfish are innervated by the tricle and bulbus. In elasmobranchs, agnatha and vagus nerve (Xth cranial nerve). Stimulation of holosteans the bulbus is replaced by the conus the vagus causes a cholinergic effect, simulating arteriosus. The conus does not increase the accel- the effects of acetylcholine, which slows the heart eration of blood, as does the bulbus in teleosts. The rate in teleosts and elasmobranchs producing blood is pumped into the ventral aorta, where bradycardia and increases the heart rate in lamprey blood pressure is in the range 4.0–7.3 kPa. After (Randall 1970). Light flashes, sudden movements passing the gills, which create ~30% of the total of objects or shadows, and touch or mechanical vi- resistance to blood flow, the blood pressure has brations may increase vagal tone. The response decreased to about 3.3–4.7 kPa in the dorsal can be blocked by injection of atropine. Some fish aorta, which flows alongside the caudal vein in also have adrenergic or stimulatory fibres from the the haemal canal formed by fusion of vertebral vagus. processes. The haemal canal thus protects the

The circulatory system may be linked to maxi- caudal vein from the pressure waves that move to mization of oxygen uptake in the gills and delivery the caudal end of the fish as the myotomes con- to the tissues. In the following I examine in more tract during swimming. The pressure for venous detail some of the key factors in this process. return is generated in the segmental veins in the muscles in the caudal region by the so-called haemal arc pump, or in the caudal sinuses or

The significance of haemoglobins

caudal pump, which can be called caudal hearts, Fish haemoglobins are mostly tetramers consist- before entering the caudal vein. Back-flow is ing of two a and two b polypeptide chains (141 and prevented by a series of valves. The caudal vein 146 amino acid residues, respectively). The basic collects blood from the cutaneous circulation, function of the haemoglobins is to ensure an ade- the posterior muscles and the remainder of the quate supply of oxygen to all parts of the organism caudal region (Satchell 1965, 1971). Hagfishes have

in which they occur. In order to accomplish this no less than five accessory hearts. For circulatory task they have developed, in the course of evolu- adaptations in air-breathing fish see Section Air tion, a common molecular mechanism based on breathing.

the principle of ligand-linked conformational

Aneural and neural mechanisms control the change in a multi-subunit structure (Wyman heart and vascular system allowing for a wide 1964). Within the framework of this common range of circulatory adjustments to environmental mechanism, however, different haemoglobins

Physiology of Living in Water

160 Antarctic fish

(ml blood per ml O

Tunas

Blood perfusion requirement

Fig. 4.5 Blood perfusion require- ments of fish in relation to blood haemoglobin concentrations.

0 0 5 10 15 (Source: based on Wood and Lenfant 1979.)

Haemoglobin concentration (g dl –1 )

have acquired special features to meet special CO 2 and nucleotide triphosphates (NTP), called needs. The significance of haemoglobins is illus- ligands (Brunori et al. 1985). Although the product trated in Fig. 4.5, which shows the impact of of the central exon of the haemoglobin gene (the haemoglobin concentration on perfusion require- oxygen-binding site) is sufficient for accomplish- ment expressed as millilitres of blood pumped by . ing the primary function of haemoglobin, the the heart (cardiac output, Q ) for each millilitre of . products of the external exons (the ligand-binding

oxygen taken up by the cells (V o 2 ) for random ex- sites) allow external modulation of the functional amples of fish species. The perfusion requirement, properties (Eaton 1982). Hence, oxygen binding at which links cardiac output and blood oxygen the haem groups is modulated to different extents transport to oxygen uptake, is calculated from the in different species by interactions with ligands flux equations shown in Section 5.1:

(Fig. 4.6). Haemoglobins exhibit a great deal of vari- ation, in terms of absolute affinity for oxygen, in

1 ˆ Vo ˙ =

Ê their susceptibility to metabolic effectors; these

2 ËÁ C aO 2 - C vO 2 ¯˜ physiological requirements of a given species. This

, (4.6) variations allow haemoglobin to fully meet the

type of tuning is primarily based on the ability of where C aO 2 -C v ¯O 2 is the difference in oxygen con- effectors to preferentially bind to one of the two tent between arterial and mixed venous blood. The quaternary conformations. In particular, preferen- Antarctic fish without haemoglobins thus have to tial binding of NTP, typically to the low oxygen pump almost ten times as much blood for the same affinity T-state, facilitates the unloading of oxygen amount of oxygen taken up as the tunas with the to the tissues (Fig. 4.6). The decrease in oxygen highest haemoglobin concentration.

affinity caused by protons is commonly referred to as the Bohr effect. When the binding of protons is so strong that the haemoglobin remains in the T-

state, in which there is no transition to the R-state Oxygen transport is based on conformational and thus no cooperativity, this is called a Root changes of the quaternary structure of the haemo- effect. This plays an important role in filling the globin molecule that are caused by the binding and swimbladder (see Fig. 4.1). Fig. 4.6 summarizes release of small solvent components, such as H + , how the oxygen uptake of fish can be modified by

Sites of control

86 Chapter 4

Blood depot and 30 erythropoietic

AV-difference

reserve

Ventilatory reserve

O 2 uptake = . AV-difference ×Q

2 content (ml dl Venous 10

Blood O

Temperature NTP PCO 2

pH

0 4 8 12 16 .

PO (kPa) Cardiac output (ml min 2 –1 ) Fig. 4.6 The magnitude of oxygen uptake of fish is illustrated as a square, where one side is the arterial–venous .

difference in blood oxygen content (AV-difference) and the other side is the cardiac output (Q ). The arterial–venous difference can be increased by increasing ventilation and/or the release of more red blood cells into the blood from the spleen (stress response), or by increasing the unloading of oxygen to the tissues under the allosteric control of ligands and temperature (the most important nucleotide triphosphates (NTPs) in fish are GTP and ATP). Cardiac output is the product of stroke volume and heartbeat frequency, and will accordingly be changed in parallel with these parameters.

regulating haemoglobin oxygen transport and car- ity of haemoglobin components appears greatest in diac output.

ectothermic animals, and in particular among fishes that may experience both rapid tidal or diur- nal, and marked seasonal, changes in ambient oxy-

gen and temperature. It has been suggested that The term ‘haemoglobin multiplicity’ means the both the number of isohaemoglobins and their occurrence of more than one haemoglobin compo- functional heterogeneity is related to the constan- nent in the same or different developmental stages cy of physicochemical conditions in the environ- of a species. Vertebrate red blood cells typically ment (Brix et al. 1999). However, demonstrating contain more than one kind of haemoglobin. this simple hypothesis has been hampered by Commonly, multiple genes are expressed that phenotypic plasticity, which can produce for code for different variants of these globins, result- example an acclimatory response of the individ- ing in ‘isohaemoglobins’. Some species show ual, and by phylogenetic divergence in physiologi- haemoglobin ‘polymorphism’, the occurrence of cal mechanisms. different ‘allohaemoglobins’ in different individ-

The significance of haemoglobin multiplicity

The selective advantages of heterogeneous uals representing different genetic strains (Brix isohaemoglobins have been considered for very et al. 1998a; Samuelsen et al. 1999). The multiplic- few, predominantly European and North Ameri-

can freshwater teleosts, including fish with atypi- cal diadromous behaviour (Brix et al. 1998b). For example, trout (Salmo trutta) and eel (Anguilla anguilla ) possess both anodic isohaemoglobins which are sensitive to temperature and pH and are characterized by strong Bohr and NTP effects, and high-affinity cathodal components with low sensi- tivity to pH and temperature (Binotti et al. 1971; Fago et al. 1995, 1997a). In addition the effects of temperature and hypoxia on the expression of spe- cific isohaemoglobins (Murad and Houston 1991; Houston and Gingras-Bedard 1994) demonstrate the phenotypic plasticity of the haemoglobin system in trout. Functionally heterogeneous isohaemoglobins of many Amazonian fishes have been suggested to be adaptive to water oxygen ten- sions in the tropics (Powers 1974, 1980; Fyhn et al. 1979; Val et al. 1990). This feature is supported by the observation that haemoglobins from ten dif- ferent species of African cichlids share an identical electrophoretic pattern and similar haemolysate oxygen-binding properties (Verheyen et al. 1986). However, the Antarctic notothenioid fishes have few isohaemoglobins present, which correlates with low activity levels and the stability of the polar marine environment (high dissolved oxygen and constant low temperature) (DiPrisco et al. 1990; DiPrisco and Tamburrini 1992). Differences in the functional properties of these simple haemoglobin systems is correlated not with envi- ronmental factors but with organismal factors such as swimming behaviour and body mass (Wells and Jokumsen 1982; Fago et al. 1997b). Haemoglobin is not expressed at all in the chan- nichthyid fishes (Cocca et al. 1997). Brix et al. (1999) showed that triplefin fishes living in shallow, thermally unstable habitats possess a greater number of cathodal isohaemoglobins, haemoglobin components that migrate towards the negative pole during electrophoresis. These species have haemoglobins with higher oxygen affinity and reduced cooperativity and which are less sensitive to changes in pH compared with haemoglobins of species occurring in more stable, deeper water habitats (Fig. 4.7). The analysis of an assemblage of closely related species circumvents some of the difficulties inherent in studies where

interpretation of experimental results is con- founded by phylogeny.