To understand the ‘osmorespiratory compromise’ (the trade-off in gill function between ion and respiratory gas exchange) during hypoxia in freshwater fish, a species-specific approach was utilized where general ionoregulatory responses to hypoxia were compared in rainbow trout (Oncorhynchus mykiss, a hypoxia-intolerant freshwater fish), and in two hypoxia-tolerant species (the goldfish Carassius auratus and the Amazonian oscar Astronotus ocellatus). In the latter two species, the dual stress situation of hypoxia plus feeding was also explored. Measurements included unidirectional and net Na^+ flux rates, ammonia excretion rates, net K^+ loss rates, branchial Na^+/K^+- ATPase and H^+- ATPase activities, and branchial morphology by scanning electron microscopy (trout and oscar only). In trout, environmental hypoxia induced complex changes in gill ionoregulatory function, where the direction and magnitude varied with both the extent and duration of the hypoxia regime. The changes in ion-regulation observed in trout in response to hypoxia indicated that the osmorespiratory compromise in this hypoxia-intolerant species was different and more complex compared to its manifestation in oscar and goldfish. This could be attributed to the adaptive physiology of the trout to oxygen-rich environments and its intolerance to low environmental oxygen availability. In both of the hypoxia-tolerant species (oscar and goldfish), there was a general reduction in gill permeability in response to severe hypoxia regardless of feeding regime, rather different from the complex patterns seen in the hypoxia-intolerant trout. However, the effects of feeding on this phenomenon differed between these species. Fed goldfish had elevated branchial fluxes that were effectively turned down during hypoxia compared to baseline flux rates maintained by starved goldfish. In contrast, fed oscars had lower fluxes compared to starved fish. Although both fed and starved fish suppressed their branchial fluxes with severe hypoxia, fed oscars delayed the turning down of fluxes. Overall, our results indicate that feeding exerts opposite effects on gill ionoregulatory function in these two hypoxia-tolerant species, and thereby differentially modulates the responses to hypoxia. These differences may relate to differences in water chemistry. Furthermore, the manifestation of the osmorespiratory compromise during hypoxia appears to be rather different from the phenomenon during exercise. / Thesis / Master of Science (MSc)

Low water pH is an ionoregulatory challenge to freshwater teleosts. Larval zebrafish (Danio rerio) exposed to pH 4 water experience increased loss of Na⁺ and respond with increases in ionocyte abundance and whole-body concentrations of cortisol. Because cortisol plays a role in regulating early development, particularly of the stress axis, the present study asked whether the increase in cortisol in embryos exposed to pH 4 water causes dysregulation of the stress axis in later life. Baseline whole-body cortisol levels measured at 4, 6, and 15 days post-fertilization (dpf) did not differ between pH 4-exposed and control fish. At 6 dpf, pH 4-exposed fish had higher concentrations of cortisol compared to control fish following a stressor, but no difference was detected at 15 dpf. In addition, transcript abundances for key genes of the stress axis did not differ between control and pH 4-exposed fish. Based on these results, exposure to pH 4 water in early life does not influence the stress axis or cortisol responses later in life. Increases in ionocyte abundance in response to low pH have the potential to alter gill morphology, thereby impairing gas transfer, a trade-off known as the osmorespiratory compromise. The present study tested the hypothesis that zebrafish reared in pH 4 water have reduced gas transfer capacity in accordance with the osmorespiratory compromise. Indicators of gas transfer and ionoregulation were measured at 6, 15, 30 and 90 dpf. Across all ages examined, fish reared in pH 4 water had significantly higher whole-body concentrations of Na⁺, higher ionocyte abundances and thicker gills than control fish. These differences were accompanied by higher ventilation frequencies and higher critical PO₂ (Pcrit) values. Additionally, adult fish raised in low pH had a significantly higher rate of oxygen consumption compared to control fish. These results support the hypothesis that development in water of low pH impairs gas transfer, as predicted by the osmorespiratory compromise.

<p>The osmorespiratory compromise is the trade-off between high gill permeability for oxygen uptake and low gill permeability for conservation of ions in fish. The fundamental purpose of this study was to examine facets of the osmorespiratory compromise in freshwater fish under conditions of extended exercise and acute hypoxia, in light of previous research identifying very different gill morphometric and ionoregulatory modifications in the hypoxia-tolerant Amazonian oscar (<em>Astronotus ocellatus</em>) and the hypoxia-intolerant rainbow trout (<em>Oncorhynchus mykiss</em>). A technique using [³H]polyethylene-4000 ([³H]PEG-4000) for branchial paracellular permeability measurement was developed, and then applied to investigate the osmorespiratory compromise during extended swimming. Methods were developed to overcome the challenges of renal [³H]PEG-4000 loss, respirometer surface adsorption, and freshwater drinking of the chemical. In both trout and oscar, corrections were employed for these sources of error – leading to findings that in both species, branchial [³H]PEG-4000 permeability was not rectified and freshwater drinking was quite high. In both species, during an 8-h swim (1.2BL/s), oxygen consumption rate increased by 75-90%; drinking rate remained high but did not increase. Branchial paracellular permeability increased by 61% during exercise in trout but remained constant in oscar. The methods developed here can be widely applied to future studies of branchial paracellular permeability.</p> <p>Unidirectional fluxes (by ²²Na) of sodium, and net fluxes of potassium, ammonia, and urea were observed during a 2-h nomoxia:2-h hypoxia (30% O₂ saturation):2-h normoxic recovery protocol – to identify adaptive trends across phylogenies and/or environments in North and South American teleosts. Strategies for coping with hypoxia appeared to be environmentally, rather than phylogenetically linked, since both the oscar (perciform) and the tambaqui (<em>Colossoma macropomum</em> – characiform) displayed characteristic permeability reduction (of apparent transcellular origin); both frequently encounter severe hypoxia in their natural habitat. Two North American perciforms, pumpkinseed (<em>Lepomis gibbosus</em>) and bluegill sunfish (<em>Lepomis macrochirus</em>) which live in less hypoxic environments, increased branchial ion leakage as in the hypoxia-intolerant trout. Four Amazonian tetra species (all characiformes: <em>Paracheirodon axelrodi, Hemigrammus rhodostomus, Moenkausia diktyota,</em> <em>Hyphessobrycon bentosi rosaceus</em>) which experience intermediate hypoxia in their native Rio Negro presented variable responses. Finally, during a 4-h swim at1.2BL/s, branchial ion fluxes were not reduced but elevated in oscar, indicating that ionoregulation in this species occurs primarily transcellularly, and that adaptive strategies to one manifestation of the osmorespiratory compromise (hypoxia) may not apply to another (exercise).</p> / Master of Science (MSc)

Goldfish undergo an adaptive morphological change in their gills involving the reversible growth and loss of a mass of cells (interlamellar cell mass, ILCM) in between the lamellae depending on oxygen demand, which can be altered by the environment or metabolic demands of the individual. The ILCM contributes to decreased passive Na+ efflux across the gill. Active uptake is maintained by the re-distribution of the ionocytes expressing Na+-uptake relevant genes (NHEs and H+-ATPase) to the outer edge of the ILCM where they can establish contact with the external environment and/or lamellar epithelium. This adaptation is thought to be partly responsible for the extreme anoxia tolerance demonstrated by goldfish, which they experience on a seasonal basis living in a pond environment. Hypoxia and hypercapnia are frequently encountered in such freshwater environments and as such, the effect of the ILCM on the capacity for acid-base regulation was evaluated. Differences in the time course of acid excretion to the environment without effect on systemic pH regulation were likely the result of the ILCM.

Goldfish undergo an adaptive morphological change in their gills involving the reversible growth and loss of a mass of cells (interlamellar cell mass, ILCM) in between the lamellae depending on oxygen demand, which can be altered by the environment or metabolic demands of the individual. The ILCM contributes to decreased passive Na+ efflux across the gill. Active uptake is maintained by the re-distribution of the ionocytes expressing Na+-uptake relevant genes (NHEs and H+-ATPase) to the outer edge of the ILCM where they can establish contact with the external environment and/or lamellar epithelium. This adaptation is thought to be partly responsible for the extreme anoxia tolerance demonstrated by goldfish, which they experience on a seasonal basis living in a pond environment. Hypoxia and hypercapnia are frequently encountered in such freshwater environments and as such, the effect of the ILCM on the capacity for acid-base regulation was evaluated. Differences in the time course of acid excretion to the environment without effect on systemic pH regulation were likely the result of the ILCM.

Goldfish undergo an adaptive morphological change in their gills involving the reversible growth and loss of a mass of cells (interlamellar cell mass, ILCM) in between the lamellae depending on oxygen demand, which can be altered by the environment or metabolic demands of the individual. The ILCM contributes to decreased passive Na+ efflux across the gill. Active uptake is maintained by the re-distribution of the ionocytes expressing Na+-uptake relevant genes (NHEs and H+-ATPase) to the outer edge of the ILCM where they can establish contact with the external environment and/or lamellar epithelium. This adaptation is thought to be partly responsible for the extreme anoxia tolerance demonstrated by goldfish, which they experience on a seasonal basis living in a pond environment. Hypoxia and hypercapnia are frequently encountered in such freshwater environments and as such, the effect of the ILCM on the capacity for acid-base regulation was evaluated. Differences in the time course of acid excretion to the environment without effect on systemic pH regulation were likely the result of the ILCM.

Goldfish undergo an adaptive morphological change in their gills involving the reversible growth and loss of a mass of cells (interlamellar cell mass, ILCM) in between the lamellae depending on oxygen demand, which can be altered by the environment or metabolic demands of the individual. The ILCM contributes to decreased passive Na+ efflux across the gill. Active uptake is maintained by the re-distribution of the ionocytes expressing Na+-uptake relevant genes (NHEs and H+-ATPase) to the outer edge of the ILCM where they can establish contact with the external environment and/or lamellar epithelium. This adaptation is thought to be partly responsible for the extreme anoxia tolerance demonstrated by goldfish, which they experience on a seasonal basis living in a pond environment. Hypoxia and hypercapnia are frequently encountered in such freshwater environments and as such, the effect of the ILCM on the capacity for acid-base regulation was evaluated. Differences in the time course of acid excretion to the environment without effect on systemic pH regulation were likely the result of the ILCM.