- Email: [email protected]
ROLE OF THE GILLS | The Osmorespiratory Compromise
The Osmorespiratory Compromise RJ Gonzalez, University of San Diego, San Diego, CA, USA ª 2011 Elsevier Inc. All rights reserved.
What Is the Osmorespiratory Compromise? The Effect of Changes in Branchial Surface Area The Effect of Changes in Branchial Diffusion Distance
Glossary Acclimation Physiological and/or biochemistry adjustments made by an organism in response to a prolonged change in their environment, e.g., temperature. Catecholamine Any of a class of amines derived from a catechol that acts as chemical messengers (a hormone or neurotransmitter). The fish’s main catecholamines are dopamine, adrenaline, and noradrenaline and these are secreted by neurons of the autonomic nervous system or specific chromaffin cells of the head kidney. Critical swimming speed (Ucrit) The Ucrit is determined in a swim tunnel where the fish are forced to swim against a current. The current velocity is increased in steps and each step is maintained for a specified time (usually 10 min to 1 h) or until the fish fatigues. Diffusion Net movement of a solute from an area of higher concentration to an area of lower concentration.
What Is the Osmorespiratory Compromise? The gill epithelium is the primary site of both gas exchange and ion regulation in fish and the term ‘osmor espiratory compromise’ refers to the functional clash that can exist between these two processes. The basis for the osmorespiratory compromise is that diffusion is involved in movement of both gases and salts (primarily Naþ and Cl�) across the branchial epithelium. Diffusion of O2 across the gill is a function of surface area, diffusion distance, mean water-to-blood PO2 difference, and O2 permeability of the epithelium. Diffusion of Naþ and Cl� across the gill depends upon surface area, diffusion distance, mean blood-to-water concentration differences, epithelial permeability to salts, and trans-epithelial potential (TEP). Thus, both gill surface area and water to-blood diffusion distance are common to the diffusion of gases and ion. In freshwater, any increase in surface area or reduction in diffusion distance to promote gas diffusion will also elevate diffusive ion loss, which must be replaced actively. Conversely, any reduction in surface area and/or
Effect of Branchial Remodeling Further Reading
Epinephrine Catecholamine hormone secreted by the adrenal gland. Hypoxia Low partial pressures of oxygen in external or internal environments. Norepinephrine Catecholamine hormone secreted by the adrenal gland. Normoxia Normal levels of oxygen in water or arterial blood. ˙ o2) Amount of oxygen Oxygen consumption (M consumed by an animal per unit time. Pcrit The environmental O2 tension below which an animal cannot sustain a routine rate of O2 uptake independent of environmental O2 tensions. The lower a fishes Pcrit, the better able it is to maintain normal levels of activity and therefore fish with lower Pcrit’s are belived to be more hypoxia tolerant than those with higher Pcrit values. Trans-epithelial potential The electrical potential across an epithelium as a function of concentration differences and relative permeabilities of all salts.
increase in diffusion distance to limit ion losses could restrict O2 uptake. The term ‘respiratory/osmoregulatory compromise’ coined in early studies eventually morphed into the slightly more user-friendly osmorespiratory com promise in the early 1990s, by which it is generally known today. The first studies of the osmorespiratory compro mise used exercise to induce changes in branchial surface area and monitored its effects on gas and/or ion transfer. Subsequent studies have used acclimation to a variety of water conditions to induce changes in branchial diffusion distance and monitored the consequences for gas and ion movement.
The Effect of Changes in Branchial Surface Area Exercise The first researchers to examine the osmorespiratory com promise argued that when fish begin to swim faster, a suite of cardiovascular and respiratory changes, mediated by catecholamine release, alter branchial blood flow in order
1389
1390 Role of the Gills | The Osmorespiratory Compromise
to promote O2 uptake. Changes include decreased vascular resistance, increased blood pressure, and re-direction of blood from more central paths to lamellar circuits, which presumably would increase exchange area (see also Gas Exchange: Respiration: An Introduction). Indeed, resting rainbow trout (Oncorhynchus mykiss) perfuse less than 60% of their gill lamellae but when given a dose of epinephrine or exposed to hypoxia, the number rises to about 75%. The implication is that during exercise, when O2 demand rises, fish increase the ‘functional surface area’ of the gills to promote O2 uptake. Such an increase in surface area, it was hypothesized, would lead to elevated rates of ion loss (primarily Naþ and Cl�). Sure enough, when rainbow trout are forced to swim by occasional prodding or electric shock, rates of appearance of radioactive 22Na in surround ing media are higher than in resting fish. Similarly, fish injected with the catecholamine norepinephrine also had higher rates of 22Na loss relative to controls or sham injected fish. Ensuing studies measured actual rates of Naþ efflux while distinguishing between short, exhaustive exercise and extended, more aerobic swimming. When fish are forced to swim exhaustively for short periods, their rates of Naþ efflux rise 70% above resting levels, confirm ing the previous findings. When both Naþ efflux and O2 uptake are measured simultaneously, additional factors are revealed to be involved in the initial high rates of ion loss. With exercise, increased O2 uptake is driven not only by the increase in functional surface area, but also by greater water-to-blood PO2 difference mainly due to higher rates of O2 consump tion in muscle tissue. The increased functional surface area to promote O2 uptake would lead to elevated rates of ion loss, but since there is no change in the blood-to-water concentration difference corresponding to the PO2 differ ence, one would expect a smaller rise in ion loss than O2 uptake. In fact, at the onset of exercise the opposite is true. Immediately after being chased for 5 min or during the first 30 min of chronic swimming at 85% Ucrit (about 4.5 body lengths s�1) O2 consumption in rainbow trout rises 2.8–3.4 times, but Naþ efflux jumps 4.5- to 5.2-fold (Ucrit – maximum prolonged swimming speed; Table 1). To put it another way, the ion to gas flux ratio rises by almost 65%. The cause of the disproportionate rise in _O) Table 1 Naþ efflux (JoutNa), rate of O2 consumption (M 2 and ion to gas flux ratio (IGR) for rainbow trout (Oncorhynchus mykiss) at rest and immediately after exhaustive exercise Treatment
JoutNa
_O M 2
IGR
At rest Post-exercise
8.8�0.8 39.7�2.3
72.2�2.2 200.7�5.4
122.2 200.0�12.3
_ O are nmol g�1 min�1, and units for IGR are Units for JoutNa and M 2 pmol Naþ/nmol O2. Values are means � SE.
Naþ efflux appears to be increased epithelial permeability to salts brought about by a catecholamine-induced increase in intra-lamellar pressure at the beginning of exercise, which stretches paracellular tight junctions (see also Osmotic, Ionic and Nitrogenous-Waste Balance: Mechanisms of Ion Transport in Freshwater Fishes). While the marked increase in branchial permeability at the onset of swimming is striking, it does not last long. During recovery from exhaustive exercise, Naþ efflux falls to within 10% of resting rates in trout while at the same time O2 consumption returns to a resting rate. Part of the reduction in ion permeability can be ascribed to lower intra-lamellar pressure due to declining catechola mine levels and/or downregulation in catecholamine receptor density, but it appears that in addition there is a significant level of control of epithelial ion permeability applied at the tight junctions. This marked ability to regulate paracellular ion permeability raises the possibi lity that during chronic swimming any increase in ion loss due to increased functional surface area could be negated by reduced permeability. Indeed, at moderate swimming speeds of 2 body lengths s�1 Naþ efflux falls to resting levels within an hour and it falls well below resting levels within 5 h. At a higher swimming speed, 4.5 body lengths s�1, Naþ efflux remains elevated well above rest ing rates for the duration of the exercise, although they drop from higher initial rates. It appears that vigorous swimming may interfere in some way with the control of ion permeability. Such high rates of Naþ efflux with vigorous exercise lead to high rates of net ion loss, which cannot be sustained for prolonged periods since they would lead to a significant depletion of internal stores and initiate a cascade of cardiovascular disturbances. The key adjustment in this case is not a control of Naþ efflux, but rather a rapid up to threefold stimulation of ion transport. The rise in active uptake allows the fish to maintain internal levels despite the higher loss rates. All work on the effects of exercise on the osmorespira tory compromise to this point had been performed on rainbow trout, which is an active, coldwater species. When other species that differ in energetic lifestyle and temperature preferences, but had similar gill sizes, were examined, significant differences were found. Resting rates of O2 consumption vary more than threefold among the species (Table 2), and the likely physiological basis for such a range is differences in branchial O2 permeability. Further, after exhaustive exercise elevation of O2 uptake for the less active species is associated with much greater rates of ion loss. For instance, after exercise banded sunfish (Enneacanthus obesus) have a rate of O2 consumption less than half that of common shiners (Notropis cornutus), but its rate of Naþ loss is more than 50% higher. Consequently, while ion to gas flux ratio for the shiner rises only 25% immediately after exercise, it is more than quadruples for the sunfish. Since shiners have
Role of the Gills | The Osmorespiratory Compromise
1391
_ O ) and ion to gas flux ratio (IGR) for common Table 2 Naþ efflux (JoutNa), rate of O2 consumption (M 2 shiners (Notropis cornutus), golden shiners (Notemigonus crysoleucas), smallmouth bass (Micropterus dolomieui), yellow perch (Perca flavescens), and banded sunfish (Enneacanthus obesus) at rest and immediately after exhaustive exercise At rest
Post-exercise
Species
JoutNa
_O M 2
IGR
JoutNa
_O M 2
IGR
Common shiner Golden shiner Smallmouth bass Yellow perch Banded sunfish
4.8�0.5 12.7�1.3 3.5�0.3 4.2�0.4 3.0�0.3
118.6�13.5 141.0�16.6 74.7�8.6 72.7�5.7 41.3�3.4
40.5 90.1 46.9 57.8 72.6
9.3�0.8 18.1�1.2 21.3�2.3 21.9�1.5 28.9�3.7
191.8�15.0 192.6�10.4 152.0�4.6 133.4�4.6 90.1�3.6
50.3�6.8 94.9�8.0 141.0�14.8 164.2�10.2 317.6�35.7
_ O are nmol g�1 min�1, and units for IGR are pmol Naþ/nmol O2. Units for JoutNa and M 2 Values are means � SE.
higher branchial O2 permeability, they can achieve higher rates of O2 uptake without the need for big changes in surface area brought about by high intra-lamellar pres sures that stimulate ion losses. Sunfish, in contrast, require much greater increases in surface area, brought about by the elevated pressure that promotes ion loss. Interestingly, species such as smallmouth bass (Micropterus dolomieu) and yellow perch (Perca flavescens), that are intermediate in resting O2 consumption, experience intermediate eleva tions in ion loss. These findings indicate that less active species such as banded sunfish, that inhabit densely vege tated coastal swamps, place less emphasis on conserving ions with activity since they probably rarely experience prolonged bouts of activity in their native habitats. The more active a fish is, the greater the potential osmotic costs. Very active species, such as the stream dwelling common (N. cornutus) and golden shiners (Notemigonus crysoleucas), avoid exorbitant osmotic costs of activity by virtue of their high branchial O2 permeability. Studies of the osmorespiratory compromise in exercis ing fish seem to confirm, in general terms, the existence of a conflict between ion and gas flux across the gills of fish based upon surface area. Such conflict is most notable when exercise is commencing and catecholamineinduced cardiovascular adjustments are occurring to enhance O2 pickup. Under those circumstances, ion loss increases more than predicted based only on functional surface area considerations due to additional elevation of branchial ion permeability. The rise in permeability is transitory and disappears within an hour or less as cate cholamines dissipate from circulation. Further, fish show considerable ability to regulate anion and cation permeability independently and can significantly lower branchial ion permeability well below resting levels and upregulate transport. Studies with a range of species indi cate that branchial gas permeability also varies among species and this influences the severity of the impact of increased gas uptake on ion loss. Fish with high O2 per meability can take up more additional O2 during exercise without much ion loss, while fish with low O2
permeability incur much higher rates of ion loss even with lower absolute rates of O2 uptake. Osmorespiratory Compromise in Seawater The basis of the osmorespiratory compromise in seawater is essentially the same as in freshwater except that the concentration differences for Naþ and Cl� are reversed (lower internally). For example, a freshwater fish typi cally has a Naþ concentration difference across the gill epithelium of around 149 mmol L�1 (inside higher), while in seawater it is over 350 mmol L�1 (outside higher), which means that increases in surface area with activity could potentially be more costly in seawater than in freshwater (see also Osmotic, Ionic and NitrogenousWaste Balance: Mechanisms of Gill Salt Secretion in Marine Teleosts). The issue is complicated, however, by the role TEP plays in salt excretion at the gills. The general model uses a positive TEP (blood relative to water) to excrete Naþ across the gills. Despite the con centration difference, Naþ would not enter across the gills because of the opposing TEP. Further complicating the issue is the fact that the gut absorbs salts in order to take up water to replace what is lost. In fact, elevated water loss with exercise is likely a bigger problem due to the ion load incurred in order to replace it. Few studies have examined the osmorespiratory com promise in seawater probably because of the technical difficulty of measuring ion fluxes in saline waters. For this reason, the few studies available have used indirect measures of ion regulation such as gill and gut Naþ/Kþ-ATPase activity and have tended to compare species that vary in energetic lifestyle and branchial surface area. More active fish have larger gills (with greater surface area) and thus should incur a greater osmoregulatory cost. Indeed, branchial Naþ/Kþ-ATPase activities of some active epipe lagic species are higher than in sluggish meso-pelagic fishes. It was argued that the sluggish fish had lower metabolic rates and thus reduced osmoregulatory costs. In contrast, branchial Naþ/Kþ-ATPase activities are not higher in the
1392 Role of the Gills | The Osmorespiratory Compromise
epipelagic yellowfin tuna (Thunnus albacares) or skipjack tuna (Katsuwonus pelamis), which are among the most aerobically active of all fish and have very large branchial surface areas, compared to less active species; it was con cluded that osmoregulatory costs are not elevated in tunas. Based on this diversity of results and paucity of studies, it is difficult to reach any conclusions of the osmorespiratory compromise in seawater fish.
The Effect of Changes in Branchial Diffusion Distance Acclimation to Soft Water For many freshwater fish, exposure to soft water (water low in Naþ and/or Ca2þ) disrupts the ability to regulate inter nal salt levels. Exposure to low [Ca2þ] water leaches Ca2þ from paracellular tight junctions and promotes diffusive ion loss, while exposure to low [Naþ] water reduces con centration-dependent active Naþ uptake. Extended time in either or both water conditions can lead to significant depletion of internal ion levels. One way to restore ion balance is to upregulate ion transport, which will either compensate for the scarcity of Naþ in the surrounding water, or counteract the higher rates of ion loss in low Ca2þ water. Upregulation of ion transport is typically associated with a proliferation of mitochondrion-rich cells (MRCs) in the branchial epithelium. In ion-rich waters, MRCs, which are much larger than the surrounding pave ment cells, are primarily found on filaments and filling-in the intra-lamellar spaces; they make up less than 10% of the surface area of the gills. As they proliferate, MRCs appear on the lamellar epithelium, which acts to raise the average water-to-blood diffusion distance for gases. Thus, acclima tion to restore ion balance in soft water could, in theory, act to impede gas exchange. Whether or not this happens in reality depends upon the degree of MRC proliferation, its effect on average water-to-blood diffusion distance, and the presence of any compensatory adjustments. When rainbow trout are exposed to soft water or treated with a combination of cortisol and ovine growth hormone, MRC density more than doubles and fractional area rises from about 5% to 20%. Visual inspection alone is enough to illustrate the impact (Figure 1). Measurements of aver age water-to-blood diffusion distance along the lamellae show that it almost doubles following MRC proliferation. This appears to be a general consequence of soft water acclimation, because similar results were obtained from the tropical Hoplias malabaricus. The proliferation of MRCs and associated increase in diffusion distance across the branchial epithelium suggests the potential for impairment of O2 uptake, but in normoxic water results vary. Some studies show a lower arterial PO2 in trout undergoing MRC proliferation, but some do not. The uptake of O2 by resting fish in normoxic waters is
Figure 1 Scanning electron micrograph of gills of rainbow trout in ion-rich water (a) and after acclimation to soft water (b). Photos by permission of S. Perry.
thought to be perfusion limited and may not be influenced by the reduction in diffusion potential. Alternatively, a compensatory elevation in ventilation frequency may be important in maintaining arterial PO2. Thickening of bran chial epithelium should also act to impair diffusion of CO2, and since CO2 excretion is thought to be diffusion limited, one would expect to see the effects of MRC proliferation in normoxic waters. Again results vary among studies. One study reported elevated arterial PCO2, while two did not. While the effects of lamellar thickening on gas exchange in normoxic waters are not completely apparent, what is clear is that thickening does have a significant effect on gas exchange when fish are challenged with hypoxia. Researchers observe reduced arterial PO2 in fish with thick ened epithelia during exposure to hypoxia. Likewise, during exercise when O2 demand is elevated, soft waterheld trout have lower Ucrit values and rely more heavily on anaerobic metabolism while swimming. It appears that as water-to-blood PO2 difference falls or O2 demand is elevated with exercise, then O2 uptake becomes diffusion limited and lamellar thickening becomes important.
Role of the Gills | The Osmorespiratory Compromise
Acclimation to soft water does not fit the original model of the osmorespiratory compromise based upon changes in functional surface area, but it does highlight other possibilities for conflict between these two pro cesses. In the original scenario, alterations to enhance diffusion of O2 for uptake also enhance diffusive ion loss. Here we see that proliferation of MRCs, to upregu late active ion transport, causes a thickening of lamella that affects O2 and CO2 diffusion. An acclimatory response to restore or defend internal ion levels is achieved at the cost of gas exchange. Acclimation to Changing Salinity Movement between freshwater and seawater requires a fish to make a host of osmoregulatory adjustments to successfully regulate internal ions. For instance, move ment from seawater to freshwater requires a hyporegulating teleost that actively excretes ions and drinks seawater (incurring an additional salt load to absorb the water) to suddenly stop drinking, start actively taking up salts, and reduce branchial ion losses. Morphological adjustments made to assist maintenance of ion balance could in theory influence gas exchange (see also Osmotic, Ionic and Nitrogenous-Waste Balance: Osmoregulation in Fishes: An Introduction). Prickly sculpins (Cottus asper), primarily a freshwater species, can still successfully and tightly regulate Naþ and Cl� in full seawater. In contrast, closely related Pacific staghorn sculpins (Leptocottus armatus) are marine and less tolerant of freshwater. Prickly sculpins have smaller gills (on a mass specific basis) than Pacific staghorn sculpins, which allows for lower ion permeability during exposure to freshwater. Further, during exposure to freshwater, prickly sculpins increase the thickness of their branchial epithelium by about 40%, which raises the water-to blood diffusion distance. This means that prickly sculpins drop an already low branchial ion permeability even further when in freshwater, thereby controlling diffusive ion loss and maintain internal levels. However, the adjust ment in diffusion distance to help conserve salts comes with a price for the prickly sculpin. Measurements of critical oxygen tensions (Pcrit) show a marked �15 torr increase in freshwater-held prickly sculpins compared to those in seawater. In other words, the increase in epithelial thickness to limit ion losses hinders O2 uptake at low environmental PO2 levels. As with acclimation to soft water, adjustments made to aid ion regulation, in turn, impede gas exchange. The morphological adjustments were different in the two cases – one a proliferation of MRC and the other a general thickening of the epithelium – both increased the water-to-blood diffusion distance for O2. In each case, differences were not obvious in normoxic waters, but became evident during exposure to hypoxic waters.
1393
Effect of Branchial Remodeling Gills of crucian carp (Carassius carassius) undergo a strik ing morphological transformation in normoxic waters at low temperature when metabolic O2 demand is low (see also Bony Fishes: Crucian Carp). Cells proliferate between branchial lamella filling in the space creating an intra-lamellar cell mass (ILCM) and greatly reducing surface area and increasing average diffusion distance With exposure to hypoxia or high temperatures, when O2 demand is elevated, these ILCMs disappear revealing the underlying lamella, restoring the higher respiratory area and reducing diffusion distance. A similar response is seen in the goldfish (Carassius auratus) and in the Lake Qinghai scaleless carp (Gymnocypris przewalskii) native to a high-altitude lake in central China. Similarly, the osteo glossid (Arapaima gigas) from the Amazon River actually loses its lamellae during ontogenetic transition from obligate water breathing at hatching to obligate air breathing (Figure 2). In this case, however, the change is irreversible. A very similar response is reported for
Figure 2 Scanning electron micrographs of gills of (a) 10 g A. gigas and (b) 1000 g A. gigas. Photos by permission of C. Brauner.
1394 Role of the Gills | The Osmorespiratory Compromise
overwintering large mouth bass (Micropterus salmoides) and for fathead minnows (Pimephales promelas) exposed to toxic water left over from refining oil sands. The explanation generally offered for such morphological changes is that a reduction in surface area and an increase in diffusion distance when O2 supplies are ample to meet demands lower osmotic costs. Results generally support the osmotic cost hypothesis. Goldfish with ILCM at low temperature have lower Cl� and polyethylene glycol (indicator of paracellular diffu sion) fluxes than goldfish at higher temperature without ILCMs. Similarly, Lake Qinghai scaleless carp that lose ILCMs in hypoxic waters experience a 10–15% drop in plasma Naþ and Cl– concentrations, suggesting higher rates of diffusive loss. In contrast, larger A. gigas that have lamellae-less gills and a fourfold greater water-to blood diffusion distance actually have diffusive ion loss rates 3 times higher than smaller fish with lamellaebearing gills. The likely difference here is probably reduced branchial ion permeability in smaller fish that more than compensates for higher surface area and shorter diffusion distance. In stark contrast to the response to hypoxia we see in Crucian carp, goldfish, and Lake Qinghai scaleless carp is the response observed in the Amazonian oscar (Astronotus ocellatus) – an extremely hypoxia-tolerant fish that can tolerate complete anoxia for up to 6 h and severe hypoxia for 1–2 days. During exposure to severe hypoxia (10–20 torr), oscars show no increase in functional gill surface area and/or reduction in diffusion distance. In fact, Naþ efflux actually drops significantly, which acts to preserve ion balance since Naþ uptake also falls due to the lack of O2 and downregulation of Naþ/Kþ-ATPase. The drop in Naþ efflux appears to be a result of pavement cells quickly covering over MRCs, thus markedly reducing their surface area and lowering branchial permeability. This is a very different take on the osmorespiratory compromise where adjustments are made to defend ion balance while not interfering with gas exchange. Perhaps conflict avoidance is only possible in such a strikingly hypoxia tolerant fish.
See also: Bony Fishes: Crucian Carp. Gas Exchange: Respiration: An Introduction. Osmotic, Ionic and Nitrogenous-Waste Balance: Mechanisms of Ion Transport in Freshwater Fishes; Osmoregulation in Fishes: An Introduction; Mechanisms of Gill Salt Secretion in Marine Teleosts.
Further Reading Booth JL (1979) The effects of oxygen supply, epinephrine and acetylcholine on the distribution of blood flow in trout gills. Journal of Experimental Biology 83: 31–39. Brill R, Swimmer Y, Taxboel C, Cousins K, and Lowe T (2001) Gill and intestinal Naþ–Kþ ATPase activity, and estimated maximal osmoregulatory costs, in three high-energy teleosts: Yellowfin tuna (Thunnus albacares), skipjack tuna (Katsuwonus pelamis), and dolphin fish (Coryphaena hippuus). Marine Biology 138: 935–944. Farrell AP, Kennedy CJ, and Kolok A (2004) Effects of wastewater from an oil-sand-refining operation on survival, hematology, gill histology, and swimming of fathead minnows. Canadian Journal of Zoology 82: 1519–1527. Gonzalez RJ and McDonald DG (1992) The relationship between oxygen consumption and ion loss in a freshwater fish. Journal of Experimental Biology 163: 317–332. Gonzalez RJ and McDonald DG (1994) The relationship between oxygen uptake and ion loss in fish from diverse habitats. Journal of Experimental Biology 190: 95–108. Henriksson P, Mandic M, and Richards JG (2008) The osmorespiratory compromise in sculpins: Impaired gas exchange is associated with freshwater tolerance. Physiological and Biochemical Zoology 81: 310–319. Nilsson GE (2007) Gill remodeling in fish – a new fashion or an ancient secret?. Journal of Experimental Biology 210: 2403–2409. Perry SF (1998) Relationships between branchial chloride cells and gas transfer in freshwater fish. Comparative Biochemistry and Physiology A 119: 9–16. Randall DJ, Baumgarten D, and Malyusz M (1972) The relationship between gas and ion transfer across the gills of fishes. Comparative Biochemistry and Physiology A 41: 629–637. Wood CM, Iftikar FI, Scott GR, et al. (2009) Regulation of gill transcellular permeability and renal function during acute hypoxia in the Amazonian oscar (Astronotus ocellatus). New angles on the osmorespiratory compromise. Journal of Experimental Biology 212: 1949–1964. Wood CM and Randall DJ (1973) The influence of swimming activity on sodium balance in the rainbow trout (Salmo gairdneri). Journal of Comparative Physiology 82: 207–233. Wood CM and Randall DJ (1973) Sodium balance in the rainbow trout (Salmo gairdneri) during extended exercise. Journal of Comparative Physiology 82: 235–256.
What Is the Osmorespiratory Compromise? The Effect of Changes in Branchial Surface Area The Effect of Changes in Branchial Diffusion Distance
Glossary Acclimation Physiological and/or biochemistry adjustments made by an organism in response to a prolonged change in their environment, e.g., temperature. Catecholamine Any of a class of amines derived from a catechol that acts as chemical messengers (a hormone or neurotransmitter). The fish’s main catecholamines are dopamine, adrenaline, and noradrenaline and these are secreted by neurons of the autonomic nervous system or specific chromaffin cells of the head kidney. Critical swimming speed (Ucrit) The Ucrit is determined in a swim tunnel where the fish are forced to swim against a current. The current velocity is increased in steps and each step is maintained for a specified time (usually 10 min to 1 h) or until the fish fatigues. Diffusion Net movement of a solute from an area of higher concentration to an area of lower concentration.
What Is the Osmorespiratory Compromise? The gill epithelium is the primary site of both gas exchange and ion regulation in fish and the term ‘osmor espiratory compromise’ refers to the functional clash that can exist between these two processes. The basis for the osmorespiratory compromise is that diffusion is involved in movement of both gases and salts (primarily Naþ and Cl�) across the branchial epithelium. Diffusion of O2 across the gill is a function of surface area, diffusion distance, mean water-to-blood PO2 difference, and O2 permeability of the epithelium. Diffusion of Naþ and Cl� across the gill depends upon surface area, diffusion distance, mean blood-to-water concentration differences, epithelial permeability to salts, and trans-epithelial potential (TEP). Thus, both gill surface area and water to-blood diffusion distance are common to the diffusion of gases and ion. In freshwater, any increase in surface area or reduction in diffusion distance to promote gas diffusion will also elevate diffusive ion loss, which must be replaced actively. Conversely, any reduction in surface area and/or
Effect of Branchial Remodeling Further Reading
Epinephrine Catecholamine hormone secreted by the adrenal gland. Hypoxia Low partial pressures of oxygen in external or internal environments. Norepinephrine Catecholamine hormone secreted by the adrenal gland. Normoxia Normal levels of oxygen in water or arterial blood. ˙ o2) Amount of oxygen Oxygen consumption (M consumed by an animal per unit time. Pcrit The environmental O2 tension below which an animal cannot sustain a routine rate of O2 uptake independent of environmental O2 tensions. The lower a fishes Pcrit, the better able it is to maintain normal levels of activity and therefore fish with lower Pcrit’s are belived to be more hypoxia tolerant than those with higher Pcrit values. Trans-epithelial potential The electrical potential across an epithelium as a function of concentration differences and relative permeabilities of all salts.
increase in diffusion distance to limit ion losses could restrict O2 uptake. The term ‘respiratory/osmoregulatory compromise’ coined in early studies eventually morphed into the slightly more user-friendly osmorespiratory com promise in the early 1990s, by which it is generally known today. The first studies of the osmorespiratory compro mise used exercise to induce changes in branchial surface area and monitored its effects on gas and/or ion transfer. Subsequent studies have used acclimation to a variety of water conditions to induce changes in branchial diffusion distance and monitored the consequences for gas and ion movement.
The Effect of Changes in Branchial Surface Area Exercise The first researchers to examine the osmorespiratory com promise argued that when fish begin to swim faster, a suite of cardiovascular and respiratory changes, mediated by catecholamine release, alter branchial blood flow in order
1389
1390 Role of the Gills | The Osmorespiratory Compromise
to promote O2 uptake. Changes include decreased vascular resistance, increased blood pressure, and re-direction of blood from more central paths to lamellar circuits, which presumably would increase exchange area (see also Gas Exchange: Respiration: An Introduction). Indeed, resting rainbow trout (Oncorhynchus mykiss) perfuse less than 60% of their gill lamellae but when given a dose of epinephrine or exposed to hypoxia, the number rises to about 75%. The implication is that during exercise, when O2 demand rises, fish increase the ‘functional surface area’ of the gills to promote O2 uptake. Such an increase in surface area, it was hypothesized, would lead to elevated rates of ion loss (primarily Naþ and Cl�). Sure enough, when rainbow trout are forced to swim by occasional prodding or electric shock, rates of appearance of radioactive 22Na in surround ing media are higher than in resting fish. Similarly, fish injected with the catecholamine norepinephrine also had higher rates of 22Na loss relative to controls or sham injected fish. Ensuing studies measured actual rates of Naþ efflux while distinguishing between short, exhaustive exercise and extended, more aerobic swimming. When fish are forced to swim exhaustively for short periods, their rates of Naþ efflux rise 70% above resting levels, confirm ing the previous findings. When both Naþ efflux and O2 uptake are measured simultaneously, additional factors are revealed to be involved in the initial high rates of ion loss. With exercise, increased O2 uptake is driven not only by the increase in functional surface area, but also by greater water-to-blood PO2 difference mainly due to higher rates of O2 consump tion in muscle tissue. The increased functional surface area to promote O2 uptake would lead to elevated rates of ion loss, but since there is no change in the blood-to-water concentration difference corresponding to the PO2 differ ence, one would expect a smaller rise in ion loss than O2 uptake. In fact, at the onset of exercise the opposite is true. Immediately after being chased for 5 min or during the first 30 min of chronic swimming at 85% Ucrit (about 4.5 body lengths s�1) O2 consumption in rainbow trout rises 2.8–3.4 times, but Naþ efflux jumps 4.5- to 5.2-fold (Ucrit – maximum prolonged swimming speed; Table 1). To put it another way, the ion to gas flux ratio rises by almost 65%. The cause of the disproportionate rise in _O) Table 1 Naþ efflux (JoutNa), rate of O2 consumption (M 2 and ion to gas flux ratio (IGR) for rainbow trout (Oncorhynchus mykiss) at rest and immediately after exhaustive exercise Treatment
JoutNa
_O M 2
IGR
At rest Post-exercise
8.8�0.8 39.7�2.3
72.2�2.2 200.7�5.4
122.2 200.0�12.3
_ O are nmol g�1 min�1, and units for IGR are Units for JoutNa and M 2 pmol Naþ/nmol O2. Values are means � SE.
Naþ efflux appears to be increased epithelial permeability to salts brought about by a catecholamine-induced increase in intra-lamellar pressure at the beginning of exercise, which stretches paracellular tight junctions (see also Osmotic, Ionic and Nitrogenous-Waste Balance: Mechanisms of Ion Transport in Freshwater Fishes). While the marked increase in branchial permeability at the onset of swimming is striking, it does not last long. During recovery from exhaustive exercise, Naþ efflux falls to within 10% of resting rates in trout while at the same time O2 consumption returns to a resting rate. Part of the reduction in ion permeability can be ascribed to lower intra-lamellar pressure due to declining catechola mine levels and/or downregulation in catecholamine receptor density, but it appears that in addition there is a significant level of control of epithelial ion permeability applied at the tight junctions. This marked ability to regulate paracellular ion permeability raises the possibi lity that during chronic swimming any increase in ion loss due to increased functional surface area could be negated by reduced permeability. Indeed, at moderate swimming speeds of 2 body lengths s�1 Naþ efflux falls to resting levels within an hour and it falls well below resting levels within 5 h. At a higher swimming speed, 4.5 body lengths s�1, Naþ efflux remains elevated well above rest ing rates for the duration of the exercise, although they drop from higher initial rates. It appears that vigorous swimming may interfere in some way with the control of ion permeability. Such high rates of Naþ efflux with vigorous exercise lead to high rates of net ion loss, which cannot be sustained for prolonged periods since they would lead to a significant depletion of internal stores and initiate a cascade of cardiovascular disturbances. The key adjustment in this case is not a control of Naþ efflux, but rather a rapid up to threefold stimulation of ion transport. The rise in active uptake allows the fish to maintain internal levels despite the higher loss rates. All work on the effects of exercise on the osmorespira tory compromise to this point had been performed on rainbow trout, which is an active, coldwater species. When other species that differ in energetic lifestyle and temperature preferences, but had similar gill sizes, were examined, significant differences were found. Resting rates of O2 consumption vary more than threefold among the species (Table 2), and the likely physiological basis for such a range is differences in branchial O2 permeability. Further, after exhaustive exercise elevation of O2 uptake for the less active species is associated with much greater rates of ion loss. For instance, after exercise banded sunfish (Enneacanthus obesus) have a rate of O2 consumption less than half that of common shiners (Notropis cornutus), but its rate of Naþ loss is more than 50% higher. Consequently, while ion to gas flux ratio for the shiner rises only 25% immediately after exercise, it is more than quadruples for the sunfish. Since shiners have
Role of the Gills | The Osmorespiratory Compromise
1391
_ O ) and ion to gas flux ratio (IGR) for common Table 2 Naþ efflux (JoutNa), rate of O2 consumption (M 2 shiners (Notropis cornutus), golden shiners (Notemigonus crysoleucas), smallmouth bass (Micropterus dolomieui), yellow perch (Perca flavescens), and banded sunfish (Enneacanthus obesus) at rest and immediately after exhaustive exercise At rest
Post-exercise
Species
JoutNa
_O M 2
IGR
JoutNa
_O M 2
IGR
Common shiner Golden shiner Smallmouth bass Yellow perch Banded sunfish
4.8�0.5 12.7�1.3 3.5�0.3 4.2�0.4 3.0�0.3
118.6�13.5 141.0�16.6 74.7�8.6 72.7�5.7 41.3�3.4
40.5 90.1 46.9 57.8 72.6
9.3�0.8 18.1�1.2 21.3�2.3 21.9�1.5 28.9�3.7
191.8�15.0 192.6�10.4 152.0�4.6 133.4�4.6 90.1�3.6
50.3�6.8 94.9�8.0 141.0�14.8 164.2�10.2 317.6�35.7
_ O are nmol g�1 min�1, and units for IGR are pmol Naþ/nmol O2. Units for JoutNa and M 2 Values are means � SE.
higher branchial O2 permeability, they can achieve higher rates of O2 uptake without the need for big changes in surface area brought about by high intra-lamellar pres sures that stimulate ion losses. Sunfish, in contrast, require much greater increases in surface area, brought about by the elevated pressure that promotes ion loss. Interestingly, species such as smallmouth bass (Micropterus dolomieu) and yellow perch (Perca flavescens), that are intermediate in resting O2 consumption, experience intermediate eleva tions in ion loss. These findings indicate that less active species such as banded sunfish, that inhabit densely vege tated coastal swamps, place less emphasis on conserving ions with activity since they probably rarely experience prolonged bouts of activity in their native habitats. The more active a fish is, the greater the potential osmotic costs. Very active species, such as the stream dwelling common (N. cornutus) and golden shiners (Notemigonus crysoleucas), avoid exorbitant osmotic costs of activity by virtue of their high branchial O2 permeability. Studies of the osmorespiratory compromise in exercis ing fish seem to confirm, in general terms, the existence of a conflict between ion and gas flux across the gills of fish based upon surface area. Such conflict is most notable when exercise is commencing and catecholamineinduced cardiovascular adjustments are occurring to enhance O2 pickup. Under those circumstances, ion loss increases more than predicted based only on functional surface area considerations due to additional elevation of branchial ion permeability. The rise in permeability is transitory and disappears within an hour or less as cate cholamines dissipate from circulation. Further, fish show considerable ability to regulate anion and cation permeability independently and can significantly lower branchial ion permeability well below resting levels and upregulate transport. Studies with a range of species indi cate that branchial gas permeability also varies among species and this influences the severity of the impact of increased gas uptake on ion loss. Fish with high O2 per meability can take up more additional O2 during exercise without much ion loss, while fish with low O2
permeability incur much higher rates of ion loss even with lower absolute rates of O2 uptake. Osmorespiratory Compromise in Seawater The basis of the osmorespiratory compromise in seawater is essentially the same as in freshwater except that the concentration differences for Naþ and Cl� are reversed (lower internally). For example, a freshwater fish typi cally has a Naþ concentration difference across the gill epithelium of around 149 mmol L�1 (inside higher), while in seawater it is over 350 mmol L�1 (outside higher), which means that increases in surface area with activity could potentially be more costly in seawater than in freshwater (see also Osmotic, Ionic and NitrogenousWaste Balance: Mechanisms of Gill Salt Secretion in Marine Teleosts). The issue is complicated, however, by the role TEP plays in salt excretion at the gills. The general model uses a positive TEP (blood relative to water) to excrete Naþ across the gills. Despite the con centration difference, Naþ would not enter across the gills because of the opposing TEP. Further complicating the issue is the fact that the gut absorbs salts in order to take up water to replace what is lost. In fact, elevated water loss with exercise is likely a bigger problem due to the ion load incurred in order to replace it. Few studies have examined the osmorespiratory com promise in seawater probably because of the technical difficulty of measuring ion fluxes in saline waters. For this reason, the few studies available have used indirect measures of ion regulation such as gill and gut Naþ/Kþ-ATPase activity and have tended to compare species that vary in energetic lifestyle and branchial surface area. More active fish have larger gills (with greater surface area) and thus should incur a greater osmoregulatory cost. Indeed, branchial Naþ/Kþ-ATPase activities of some active epipe lagic species are higher than in sluggish meso-pelagic fishes. It was argued that the sluggish fish had lower metabolic rates and thus reduced osmoregulatory costs. In contrast, branchial Naþ/Kþ-ATPase activities are not higher in the
1392 Role of the Gills | The Osmorespiratory Compromise
epipelagic yellowfin tuna (Thunnus albacares) or skipjack tuna (Katsuwonus pelamis), which are among the most aerobically active of all fish and have very large branchial surface areas, compared to less active species; it was con cluded that osmoregulatory costs are not elevated in tunas. Based on this diversity of results and paucity of studies, it is difficult to reach any conclusions of the osmorespiratory compromise in seawater fish.
The Effect of Changes in Branchial Diffusion Distance Acclimation to Soft Water For many freshwater fish, exposure to soft water (water low in Naþ and/or Ca2þ) disrupts the ability to regulate inter nal salt levels. Exposure to low [Ca2þ] water leaches Ca2þ from paracellular tight junctions and promotes diffusive ion loss, while exposure to low [Naþ] water reduces con centration-dependent active Naþ uptake. Extended time in either or both water conditions can lead to significant depletion of internal ion levels. One way to restore ion balance is to upregulate ion transport, which will either compensate for the scarcity of Naþ in the surrounding water, or counteract the higher rates of ion loss in low Ca2þ water. Upregulation of ion transport is typically associated with a proliferation of mitochondrion-rich cells (MRCs) in the branchial epithelium. In ion-rich waters, MRCs, which are much larger than the surrounding pave ment cells, are primarily found on filaments and filling-in the intra-lamellar spaces; they make up less than 10% of the surface area of the gills. As they proliferate, MRCs appear on the lamellar epithelium, which acts to raise the average water-to-blood diffusion distance for gases. Thus, acclima tion to restore ion balance in soft water could, in theory, act to impede gas exchange. Whether or not this happens in reality depends upon the degree of MRC proliferation, its effect on average water-to-blood diffusion distance, and the presence of any compensatory adjustments. When rainbow trout are exposed to soft water or treated with a combination of cortisol and ovine growth hormone, MRC density more than doubles and fractional area rises from about 5% to 20%. Visual inspection alone is enough to illustrate the impact (Figure 1). Measurements of aver age water-to-blood diffusion distance along the lamellae show that it almost doubles following MRC proliferation. This appears to be a general consequence of soft water acclimation, because similar results were obtained from the tropical Hoplias malabaricus. The proliferation of MRCs and associated increase in diffusion distance across the branchial epithelium suggests the potential for impairment of O2 uptake, but in normoxic water results vary. Some studies show a lower arterial PO2 in trout undergoing MRC proliferation, but some do not. The uptake of O2 by resting fish in normoxic waters is
Figure 1 Scanning electron micrograph of gills of rainbow trout in ion-rich water (a) and after acclimation to soft water (b). Photos by permission of S. Perry.
thought to be perfusion limited and may not be influenced by the reduction in diffusion potential. Alternatively, a compensatory elevation in ventilation frequency may be important in maintaining arterial PO2. Thickening of bran chial epithelium should also act to impair diffusion of CO2, and since CO2 excretion is thought to be diffusion limited, one would expect to see the effects of MRC proliferation in normoxic waters. Again results vary among studies. One study reported elevated arterial PCO2, while two did not. While the effects of lamellar thickening on gas exchange in normoxic waters are not completely apparent, what is clear is that thickening does have a significant effect on gas exchange when fish are challenged with hypoxia. Researchers observe reduced arterial PO2 in fish with thick ened epithelia during exposure to hypoxia. Likewise, during exercise when O2 demand is elevated, soft waterheld trout have lower Ucrit values and rely more heavily on anaerobic metabolism while swimming. It appears that as water-to-blood PO2 difference falls or O2 demand is elevated with exercise, then O2 uptake becomes diffusion limited and lamellar thickening becomes important.
Role of the Gills | The Osmorespiratory Compromise
Acclimation to soft water does not fit the original model of the osmorespiratory compromise based upon changes in functional surface area, but it does highlight other possibilities for conflict between these two pro cesses. In the original scenario, alterations to enhance diffusion of O2 for uptake also enhance diffusive ion loss. Here we see that proliferation of MRCs, to upregu late active ion transport, causes a thickening of lamella that affects O2 and CO2 diffusion. An acclimatory response to restore or defend internal ion levels is achieved at the cost of gas exchange. Acclimation to Changing Salinity Movement between freshwater and seawater requires a fish to make a host of osmoregulatory adjustments to successfully regulate internal ions. For instance, move ment from seawater to freshwater requires a hyporegulating teleost that actively excretes ions and drinks seawater (incurring an additional salt load to absorb the water) to suddenly stop drinking, start actively taking up salts, and reduce branchial ion losses. Morphological adjustments made to assist maintenance of ion balance could in theory influence gas exchange (see also Osmotic, Ionic and Nitrogenous-Waste Balance: Osmoregulation in Fishes: An Introduction). Prickly sculpins (Cottus asper), primarily a freshwater species, can still successfully and tightly regulate Naþ and Cl� in full seawater. In contrast, closely related Pacific staghorn sculpins (Leptocottus armatus) are marine and less tolerant of freshwater. Prickly sculpins have smaller gills (on a mass specific basis) than Pacific staghorn sculpins, which allows for lower ion permeability during exposure to freshwater. Further, during exposure to freshwater, prickly sculpins increase the thickness of their branchial epithelium by about 40%, which raises the water-to blood diffusion distance. This means that prickly sculpins drop an already low branchial ion permeability even further when in freshwater, thereby controlling diffusive ion loss and maintain internal levels. However, the adjust ment in diffusion distance to help conserve salts comes with a price for the prickly sculpin. Measurements of critical oxygen tensions (Pcrit) show a marked �15 torr increase in freshwater-held prickly sculpins compared to those in seawater. In other words, the increase in epithelial thickness to limit ion losses hinders O2 uptake at low environmental PO2 levels. As with acclimation to soft water, adjustments made to aid ion regulation, in turn, impede gas exchange. The morphological adjustments were different in the two cases – one a proliferation of MRC and the other a general thickening of the epithelium – both increased the water-to-blood diffusion distance for O2. In each case, differences were not obvious in normoxic waters, but became evident during exposure to hypoxic waters.
1393
Effect of Branchial Remodeling Gills of crucian carp (Carassius carassius) undergo a strik ing morphological transformation in normoxic waters at low temperature when metabolic O2 demand is low (see also Bony Fishes: Crucian Carp). Cells proliferate between branchial lamella filling in the space creating an intra-lamellar cell mass (ILCM) and greatly reducing surface area and increasing average diffusion distance With exposure to hypoxia or high temperatures, when O2 demand is elevated, these ILCMs disappear revealing the underlying lamella, restoring the higher respiratory area and reducing diffusion distance. A similar response is seen in the goldfish (Carassius auratus) and in the Lake Qinghai scaleless carp (Gymnocypris przewalskii) native to a high-altitude lake in central China. Similarly, the osteo glossid (Arapaima gigas) from the Amazon River actually loses its lamellae during ontogenetic transition from obligate water breathing at hatching to obligate air breathing (Figure 2). In this case, however, the change is irreversible. A very similar response is reported for
Figure 2 Scanning electron micrographs of gills of (a) 10 g A. gigas and (b) 1000 g A. gigas. Photos by permission of C. Brauner.
1394 Role of the Gills | The Osmorespiratory Compromise
overwintering large mouth bass (Micropterus salmoides) and for fathead minnows (Pimephales promelas) exposed to toxic water left over from refining oil sands. The explanation generally offered for such morphological changes is that a reduction in surface area and an increase in diffusion distance when O2 supplies are ample to meet demands lower osmotic costs. Results generally support the osmotic cost hypothesis. Goldfish with ILCM at low temperature have lower Cl� and polyethylene glycol (indicator of paracellular diffu sion) fluxes than goldfish at higher temperature without ILCMs. Similarly, Lake Qinghai scaleless carp that lose ILCMs in hypoxic waters experience a 10–15% drop in plasma Naþ and Cl– concentrations, suggesting higher rates of diffusive loss. In contrast, larger A. gigas that have lamellae-less gills and a fourfold greater water-to blood diffusion distance actually have diffusive ion loss rates 3 times higher than smaller fish with lamellaebearing gills. The likely difference here is probably reduced branchial ion permeability in smaller fish that more than compensates for higher surface area and shorter diffusion distance. In stark contrast to the response to hypoxia we see in Crucian carp, goldfish, and Lake Qinghai scaleless carp is the response observed in the Amazonian oscar (Astronotus ocellatus) – an extremely hypoxia-tolerant fish that can tolerate complete anoxia for up to 6 h and severe hypoxia for 1–2 days. During exposure to severe hypoxia (10–20 torr), oscars show no increase in functional gill surface area and/or reduction in diffusion distance. In fact, Naþ efflux actually drops significantly, which acts to preserve ion balance since Naþ uptake also falls due to the lack of O2 and downregulation of Naþ/Kþ-ATPase. The drop in Naþ efflux appears to be a result of pavement cells quickly covering over MRCs, thus markedly reducing their surface area and lowering branchial permeability. This is a very different take on the osmorespiratory compromise where adjustments are made to defend ion balance while not interfering with gas exchange. Perhaps conflict avoidance is only possible in such a strikingly hypoxia tolerant fish.
See also: Bony Fishes: Crucian Carp. Gas Exchange: Respiration: An Introduction. Osmotic, Ionic and Nitrogenous-Waste Balance: Mechanisms of Ion Transport in Freshwater Fishes; Osmoregulation in Fishes: An Introduction; Mechanisms of Gill Salt Secretion in Marine Teleosts.
Further Reading Booth JL (1979) The effects of oxygen supply, epinephrine and acetylcholine on the distribution of blood flow in trout gills. Journal of Experimental Biology 83: 31–39. Brill R, Swimmer Y, Taxboel C, Cousins K, and Lowe T (2001) Gill and intestinal Naþ–Kþ ATPase activity, and estimated maximal osmoregulatory costs, in three high-energy teleosts: Yellowfin tuna (Thunnus albacares), skipjack tuna (Katsuwonus pelamis), and dolphin fish (Coryphaena hippuus). Marine Biology 138: 935–944. Farrell AP, Kennedy CJ, and Kolok A (2004) Effects of wastewater from an oil-sand-refining operation on survival, hematology, gill histology, and swimming of fathead minnows. Canadian Journal of Zoology 82: 1519–1527. Gonzalez RJ and McDonald DG (1992) The relationship between oxygen consumption and ion loss in a freshwater fish. Journal of Experimental Biology 163: 317–332. Gonzalez RJ and McDonald DG (1994) The relationship between oxygen uptake and ion loss in fish from diverse habitats. Journal of Experimental Biology 190: 95–108. Henriksson P, Mandic M, and Richards JG (2008) The osmorespiratory compromise in sculpins: Impaired gas exchange is associated with freshwater tolerance. Physiological and Biochemical Zoology 81: 310–319. Nilsson GE (2007) Gill remodeling in fish – a new fashion or an ancient secret?. Journal of Experimental Biology 210: 2403–2409. Perry SF (1998) Relationships between branchial chloride cells and gas transfer in freshwater fish. Comparative Biochemistry and Physiology A 119: 9–16. Randall DJ, Baumgarten D, and Malyusz M (1972) The relationship between gas and ion transfer across the gills of fishes. Comparative Biochemistry and Physiology A 41: 629–637. Wood CM, Iftikar FI, Scott GR, et al. (2009) Regulation of gill transcellular permeability and renal function during acute hypoxia in the Amazonian oscar (Astronotus ocellatus). New angles on the osmorespiratory compromise. Journal of Experimental Biology 212: 1949–1964. Wood CM and Randall DJ (1973) The influence of swimming activity on sodium balance in the rainbow trout (Salmo gairdneri). Journal of Comparative Physiology 82: 207–233. Wood CM and Randall DJ (1973) Sodium balance in the rainbow trout (Salmo gairdneri) during extended exercise. Journal of Comparative Physiology 82: 235–256.