Kathleen M. Gilmour

Acid–base balance and CO2 excretion in fish: Unanswered questions and emerging models

Carbon dioxide (CO(2)) excretion and acid-base regulation in fish are linked, as in other animals, though the reversible reactions of CO(2) and the acid-base equivalents H(+) and HCO(3)(-): CO(2)+H(2)O<-->H(+)+HCO(3)(-). These relationships offer two potential routes through which acid-base disturbances may be regulated. Respiratory compensation involves manipulation of ventilation so as to retain CO(2) or enhance CO(2) loss, with the concomitant readjustment of the CO(2) reaction equilibrium and the resultant changes in H(+) levels. In metabolic compensation, rates of direct H(+) and HCO(3)(-) exchange with the environment are manipulated to achieve the required regulation of pH; in this case, hydration of CO(2) yields the necessary H(+) and HCO(3)(-) for exchange. Because ventilation in fish is keyed primarily to the demands of extracting O(2) from a medium of low O(2) content, the capacity to utilize respiratory compensation of acid-base disturbances is limited and metabolic compensation across the gill is the primary mechanism for re-establishing pH balance. The contribution of branchial acid-base exchanges to pH compensation is widely recognized, but the molecular mechanisms underlying these exchanges remain unclear. The relatively recent application of molecular approaches to this question is generating data, sometimes conflicting, from which models of branchial acid-base exchange are gradually emerging. The critical importance of the gill in acid-base compensation in fish, however, has made it easy to overlook other potential contributors. Recently, attention has been focused on the role of the kidney and particularly the molecular mechanisms responsible for HCO(3)(-) reabsorption. It is becoming apparent that, at least in freshwater fish, the responses of the kidney are both flexible and essential to complement the role of the gill in metabolic compensation. Finally, while respiratory compensation in fish is usually discounted, the few studies that have thoroughly characterized ventilatory responses during acid-base disturbances in fish suggest that breathing may, in fact, be adjusted in response to pH imbalances. How this is accomplished and the role it plays in re-establishing acid-base balance are questions that remain to be answered.

Physiological Causes and Consequences of Social Status in Salmonid Fish

Abstract Social interactions in small groups of juvenile rainbow trout (Oncorhynchus mykiss) lead to the formation of dominance hierarchies. Dominant fish hold better positions in the environment, gain a larger share of the available food and exhibit aggression towards fish lower in the hierarchy. By contrast, subordinate fish exhibit behavioural inhibition, including reduced activity and feeding. The behavioural characteristics associated with social status are likely the result of changes in brain monoamines resulting from social interactions. Whereas substantial physiological benefits, including higher growth rates and condition factor, are experienced by dominant trout, low social status appears to be a chronic stress, as indicated by sustained elevation of circulating cortisol concentrations in subordinate fish. High cortisol levels, in turn, may be responsible for many of the deleterious physiological consequences of low social status, including lower growth rates and condition factor, immunosuppression and increased mortality. Circulating cortisol levels may also be a factor in determining the outcome of social interactions in pairs of rainbow trout, and hence in determining social status. Rainbow trout treated with cortisol were significantly more likely to become subordinate in paired encounters with smaller untreated conspecifics.

Plasma Cortisol Concentrations Before and After Social Stress in Rainbow Trout and Brown Trout

Two related experiments examined the relationship between plasma cortisol concentrations and the development of social hierarchies in fish. In the first, rainbow trout, Oncorhynchus mykiss, and brown trout, Salmo trutta, were observed for dominance interactions when confined within single‐species pairs for 4, 48, or 168 h. Subordinate members of a pair exhibited significantly higher cortisol concentrations than dominant and single fish, but the pattern of cortisol elevation differed between the two species, being quicker to rise and increasing to a higher level in rainbow trout. Cortisol concentrations were correlated with behavioural measurements; the more subordinate the behaviour exhibited by a fish, the higher its cortisol concentration. Social stress was a chronic stressor, and no acclimation to social status occurred during the week. In the second experiment, measurements of plasma cortisol were made before pairing of rainbow trout and then after 48 h of confinement in pairs. Subordinate fish demonstrated significantly higher concentrations of plasma cortisol both before and after social stress. It therefore appears that in addition to cortisol being elevated during periods of social stress, an association may exist between initial cortisol levels and the likelihood of a fish becoming subordinate.

Corticotropin-releasing factor and neuropeptide Y mRNA levels are elevated in the preoptic area of socially subordinate rainbow trout

The objectives of this study were to characterize rainbow trout (Oncorhynchus mykiss) corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) cDNAs and to determine their mRNA levels in response to social stress. Standard cloning techniques were used to obtain cDNAs, sequences for trout NPY and two CRF isoforms. At the predicted amino acid level, our NPY sequence differs from the trout amino acid sequence reported by. A phylogenetic analysis suggests that the two CRF isoforms result from a gene duplication that occurred in a common ancestor of salmonids. A tissue distribution demonstrated that the mRNAs of both CRF isoforms are predominantly present in the preoptic area of the trout brain, whereas NPY mRNA is more abundant in the telencephalon. Pairs of sized-matched juvenile female trout were allowed to interact for 72 h and social ranks were assigned on the basis of behavioural observations. Mean plasma cortisol levels were 13-fold higher in subordinate than in dominant trout. As measured by ribonuclease protection assay, CRF1 and NPY mRNA levels were respectively 51 and 32% higher in the preoptic area of subordinate trout; in addition, CRF1 and NPY mRNA levels were positively correlated (R2=0.44). These results suggest that subordinate rainbow trout chronically maintain high levels of CRF mRNA during social stress and that NPY may be involved in the control of the stress axis in trout.

The effects of cortisol administration on social status and brain monoaminergic activity in rainbow trout Oncorhynchus mykiss

SUMMARY The hypothesis that circulating cortisol levels influence the outcome of social interactions in rainbow trout was tested. Juvenile rainbow trout Oncorhynchus mykiss were given a single intraperitoneal (i.p.) implant containing either cortisol (110 mg kg–1 fish), or cortisol plus the glucocorticoid receptor antagonist RU486 (mifepristone; 1100 mg kg–1 fish), and sampled after 5 days of social interactions with either a similar sized (<1.5% difference in fork length) or smaller conspecific (>5% difference). Within size-matched pairs of fish, cortisol treatment significantly increased the probability that the treated fish within each pair became subordinate, an effect that was abolished by simultaneous administration of RU486. Cortisol treatment also reduced the usual success of the larger fish within a pair to preferentially become dominant from 86% to 40% of pairs. To investigate one potential mechanism underlying the apparent effect of cortisol in predisposing trout to low social status, fish were treated with cortisol or cortisol+RU486 for 5 days, after which brain monoamines [5-hydroxytryptamine (5-HT); dopamine (DA)] and their major metabolites [5-hydroxyindolacetic acid (5-HIAA); 3,4-dihydroxy-phenylacetic acid (DOPAC)] were measured. Significant increases of serotonergic activity ([5-HIAA]/[5-HT] ratio) were detected in the telencephalon with cortisol treatment, an effect that was eliminated by simultaneous administration of RU486. Also, cortisol treatment significantly decreased dopaminergic activity in the telencephalon. Somewhat surprisingly, the effects of cortisol treatment on monoaminergic activity in the hypothalamus were opposite to those in the telencephalon. Moreover, in no case did administration of RU486 abolish these effects. These results suggest that the effects of cortisol on social status in rainbow trout may be mediated via the modulation of central signaling systems and subsequent changes in behaviour and/or competitive ability, although the exact site of action in the brain remains uncertain.

Carbonic anhydrase and acid–base regulation in fish

SUMMARY Carbonic anhydrase (CA) is the zinc metalloenzyme that catalyses the reversible reactions of CO2 with water. CA plays a crucial role in systemic acid–base regulation in fish by providing acid–base equivalents for exchange with the environment. Unlike air-breathing vertebrates, which frequently utilize alterations of breathing (respiratory compensation) to regulate acid–base status, acid–base balance in fish relies almost entirely upon the direct exchange of acid–base equivalents with the environment (metabolic compensation). The gill is the critical site of metabolic compensation, with the kidney playing a supporting role. At the gill, cytosolic CA catalyses the hydration of CO2 to H+ and HCO3– for export to the water. In the kidney, cytosolic and membrane-bound CA isoforms have been implicated in HCO3– reabsorption and urine acidification. In this review, the CA isoforms that have been identified to date in fish will be discussed together with their tissue localizations and roles in systemic acid–base regulation.

The CO2/pH ventilatory drive in fish ☆

That ventilation in fish is driven by O2 has long been accepted. The O2 ventilatory drive reflects the much lower capacitance of water for O2 than for CO2, and is mediated by O2 receptors that are distributed throughout the gill arches and that monitor both internal and external O2 levels. In recent years, however, evidence has amassed in support of the existence of a ventilatory drive in fish that is keyed to CO2 and/or pH. While ventilatory responses to CO2/pH may be mediated in part by the O2 drive through CO2/pH-induced changes in blood O2 status, CO2/pH also appear to stimulate ventilation directly. The receptors involved in this pathway are as yet unknown, but the experimental evidence available to date supports the involvement of branchial CO2-sensitive chemoreceptors with an external orientation. Internally-oriented CO2-sensitive chemoreceptors may also be involved, although evidence on this point remains equivocal. In the present paper, the evidence for a CO2/pH-keyed ventilatory drive in fish will be reviewed.

Physiological effects of dominance hierarchies within groups of brown trout, Salmo trutta, held under simulated natural conditions

While the existence of dominance hierarchies within natural populations of salmonids is well known, little is known about the physiological consequences of these social interactions. To investigate such physiological effects, replicate groups of four brown trout (Salmo trutta) were held under simulated natural conditions in an artificial stream tank. Behavioural observations allowed the fish to be ranked for dominance. After two weeks, physiological status was assessed through measurements of specific growth rate, condition factor, plasma cortisol and ion concentrations, haematocrit, leucocrit, hepatosomatic index, hepatic glycogen concentration, interrenal cell nuclear area and gill epithelium chloride cell density. Weight gain in the first-ranking (dominant) fish was significantly higher than in the second-ranking fish. In addition, the condition factor of the second-ranking fish decreased over the experimental period while those of the first- and third- ranking fish increased, resulting in significant differences among the three groups. The only other physiological parameter which varied significantly among the ranked fish was chloride cell density, which was significantly higher in the second-ranking fish than in the dominant fish. Cortisol concentrations were low in all fish and did not vary significantly with dominance status. Overall, the least beneficial position, in physiological terms, appears to be the second rank in the dominance hierarchy.

Perspectives on carbonic anhydrase

In the years since Larimer and Schmidt-Nielsen published their examination of red blood cell (RBC) carbonic anhydrase (CA) activities as a function of body mass in mammals, our knowledge of CA has expanded dramatically. We are now aware of the diversity of CA isoforms and their implication in a wide array of physiological processes. The catalytic mechanism of CA has been described, and numerous compounds that function as activators or inhibitors of CA activity have been identified. CA is investigated as a diagnostic tumor marker, and CA inhibitors are used or emerging as clinical treatments for diseases as diverse as glaucoma, cancer and obesity. Yet despite the intensity of research effort over the last 50years and the wealth of information that has accumulated, the questions asked by Larimer and Schmidt-Nielsen remain relevant today - we still have much to learn about the patterns and physiological significance of interspecific differences in CA expression and activity.

Effects of an environmental perturbation on the social behaviour and physiological function of brown trout

We investigated the effect of an environmental perturbation on brown trout, Salmo trutta, dominance hierarchies. Hierarchies were established over a 1-week period under constant simulated natural conditions in artificial stream tanks. In the perturbation treatment water levels were then lowered for a week to simulate a drought, whereas conditions remained the same in the control tanks. We recorded behavioural interactions before and after the environmental perturbation. After the 2-week experiment, we killed the fish and measured growth rate, plasma cortisol, hepatic glycogen content, hepatosomatic index, gill epithelial chloride cell densities and interrenal cell nuclear areas. Aggression showed a nonsignificant increase in the drought tanks when the water level was lowered, and behaviour and social ranking of the fish were significantly affected by the environmental perturbation with a general breakdown in the social hierarchy. The pronounced benefits of dominance in terms of growth rate observed in the control tanks were not apparent in the drought tanks. However, the cortisol concentrations of the drought fish were not significantly higher than those of control fish at the end of the experiment, suggesting that the environmental change itself was not physiologically stressful in the long term. Neither were any other physiological parameters measured significantly different to those of the control tanks. Given that a stable social system (and its physiological consequences) was observed only in a constant environment, misleading conclusions may be drawn if environmental perturbations are not incorporated into experiments studying the behaviour of stream-living fish in simulated natural conditions.