Salmonid fishes have been reported to have a remarkable ontogeny of cone photoreceptors in their retina. The ultraviolet-sensitive (UVS) cones are of particular interest, as they disappear from, and reappear into, the retina. These events occur at times associated with migration to marine waters, and the return migration to freshwater spawning grounds, respectively. The primary goal of this thesis was to discover the mechanisms underlying this ontogeny of UVS cones by studying a salmonid, the rainbow trout (Oncorhynchus mykiss). Two hypotheses were considered: 1) UVS cones become dormant, similar to speculations regarding light damage of rod photoreceptors in albino trout; 2) UVS cones die and subsequently regenerate from stem cells known to robustly proliferate in trout retina. I cloned partial cDNAs of each opsin from trout and used them to develop in sjtu hybridization labelling of photoreceptors. I introduced the ability to assess UV sensitivity utilizing electroretinograms, and developed a polyclonal antibody against the UVS opsin, to label UVS cones in immunohistochemistry. I combined these tools to examine trout UVS cones during natural development, and found that it was similar to events during thyroid hormone (TH) treatment. I used labels and inhibitors of programmed cell death to determine that UVS cone death is a major mechanism of UVS cone disappearance. UVS cones reappeared into the retina following termination of TH treatment. Application of cell fate markers indicates that reappearing UVS cones can be generated from proliferating stem cells. Electroretinograms demonstrated that these regenerated UVS cones sufficiently integrate into the retina to pass signals onto second order neurons. This represents the only known example of cone photoreceptors regenerating from stem cells during natural development. I speculate on the adaptive value of the ontogeny of UVS cones. I also investigated mechanisms underlying the apparent survival of rod photoreceptors when albino trout retina receive light-induced damage. Previous conclusions in this area had been influential in forming the hypotheses of UVS cone ontogeny. Two hypotheses were envisioned: 1) rod photoreceptors were surviving light damage; 2) rods were being killed by light but quickly replaced by proliferating retinal cells. My results support the latter hypothesis.

Graduation date: 1994

From Introduction: Rainbow trout (Salmo gairdneri) were introduced into Europe from North America in the latter half of the last century. They can tolerate higher water temperatures and lower oxygen concentrations than the native brown trout (Salmo trutta). Rainbows grow faster than brown trout under similar conditions and are thus particularly attractive for artificial rearing methods. In Denmark there is a thriving rainbow trout farming industry producing about 9,000 metric tons annually which is largely exported for table use (Mills 1971). In Britain production of rainbow trout for food is not on such a large scale but they form the basis of a considerable sport fishery. In Scotland and Northern England although rainbow trout reach sexual maturity they do not generally breed so the population is entirely dependent on restocking with hatchery reared fish, thus although living more or less wild in many British waters , this species is essentially an artificially managed resource upon which man can impose genetic selection (Donaldson and Olson 1957) as well as normal fishery controls. A detailed understanding of the biology and physiology of this species is hence of particular importance.

Graduation date: 1993

The inter-tissue metabolic responses to hypoxia were determined in lungfish (Protopterus aethiopicus), and trout (Salmo gairdneri). Lungfish respond to hypoxia with a reduction in metabolic rate. It was intended to determine which tissue, or tissues exhibit decreased metabolic rates during hypoxia, and then compare the results with the metabolic reactions observed in trout, which are not reported to reduce metabolic rate during hypoxia. The metabolic potentials of the heart, brain, white muscle and liver in the African lungfish were estimated using enzymatic data. Metabolic effects of a 12 hr submergence were monitored using metabolite measurements. Heart was the most oxidative tissue, but also showed the greatest anaerobic potential. The brain displayed relatively low oxidative capabilities. White muscle remained almost inert. Although high energy phosphate concentrations in brain and heart did not fall during submergence, glycolysis was activated as indicated by cross-over plots, depletion of endogenous glycogen stores, and lactate accumulation. Blood-tissue lactate and glucose gradients indicated (1) that the heart and brain released lactate throughout submergence, (2) that after 12 hr of submersion the brain and heart were probably obtaining all their required glucose from the blood (3) that the liver released glucose throughout submergence, and (4) the white muscle was metabolically isolated from the rest of the body during submergence. The lack of measurable changes in white muscle metabolite concentrations coupled with the low enzyme activities leads to the suggestion that the most significant adaptation to hypoxia in these fishes may not be the capacity for increased anaerobic energy production. Instead, it is likely that the ability of the muscle to prevent the activation of glycolysis during hypoxic dysoxia is the key to the animal's survival. Histochemical and ultrastructural studies were done on the axial musculature of the lungfish. The small wedge of red coloured muscle evident upon gross examination was shown by histochemical demonstrations of lactate and succinate dehydrogenases, of adenosine triphosphatases, and of lipid to be composed of a mosaic of red and intermediate fibres. Respectively, these fibres measured 23.6 and 34.3 microns in average diameter. The bulk of the myotome is composed of white fibres having an average diameter of 67.3 microns. Mitochondrial density, capillarity and lipid content were very low for all fibres. These data suggest that the axial musculature is geared primarily for anaerobic function. The relatively large percentage of white muscle indicates that the overall metabolic rate of the axial muscle is low. The capacity of the muscle to exist with a reduced rate of ATP turnover (as was suggested above) may be related to the large proportion of white fibres present in the myotome. Tissue metabolites were measured in a hypoxia sensitive organism, the Rainbow trout (Salmo qairdneri), before and after exposure for 3 hr to inspired oxygen tensions of 20 torr (at 4°C). There were small changes in the brain but the energy status was maintained. The red muscle was the least affected. White muscle creatine phosphate was depleted. Various data indicate that the white muscle is the major user of glycolytic substrates and the major producer of lactate. The heart is stressed as indicated by a decline in glycogen, ATP, CrP, and the total adenylate pool. The liver exhibited declines in every indicator of metabolic homeostasis. The liver concentrations of glycogen did not decline. The fact that anaerobic glycolysis has been activated in the white muscle, while the muscle remains in metabolic communication with the other tissues via the blood, supports the suggestion that the metabolism of the white muscle will have a pronounced effect on the metabolic status of the whole animal. The trout is maintaining its rate of oxygen uptake while activating anaerobic glycolysis in the attempt to maintain 'normal' rates of energy utilization. The turnover rates of glucose and lactate were measured in trout subjected to the same hypoxic stress as above. Glucose turnover did not change while lactate turnover increased from 2.8 ± 0.4 µmoles/min./kg to 20.6 ± 6.8 µmoles/min./kg. The lack of increase in glucose turnover was attributed to the observation that liver glycogen concentrations do not change and so there is no increase in glucose flux. The increase in lactate turnover emphasizes the fact that anaerobic glycolysis is activated and that some tissues are oxidizing lactate. The problem of when a cell becomes hypoxic and the reactions of the cell to that stress is addressed. The cell (tissue, organ, animal) has two options if oxygen supply drops to a level which prevents oxidative metabolism from supplying all of the requirements for ATP synthesis. The cell may exhibit a decline in requirements, in which case the rate of ATP production need not be as high as in the oxidative state or, conversely, anaerobic energy production may increase in the attempt to maintain ATP production rates. The lungfish muscle appears to be capable of the former, thus preserving substrates for other tissues and reducing the rate of end-product formation. The trout white muscle, on the other hand, exerts a major influence upon the other tissues when the animal is stressed with hypoxia. The term 'energy conformer' is applied to animals which do not maintain oxygen uptake in the face of a declining supply, and which allow ATP production to decline concomittantly by not activating glycolysis to a marked degree. An energy regulator would activate glycolysis in the attempt to maintain oxidative rates of ATP production. The trout is more of an energy regulator than is the lungfish with the main difference in this capacity being in the white muscle. / Science, Faculty of / Zoology, Department of / Graduate

The peripheral-type benzodiazepine receptor (PTBR) of trout brain was pharmacologically characterized and pyrethroid interaction with this site investigated. High-affinity binding sites for [³H]PK 11195 were detected in brain membranes of rainbow trout; these shared some of the characteristics of the PTBR of rodent brain (i.e., high affinity for PK 11195 and an endogenous ligand protoporphyrin IX) but were unique in the low affinity for Ro5-4864. Permethrin displaced [³H]PK 11195 binding with micromolar affinity while deltamethrin had less than 50% efficacy at displacement. Thus the PTBR appeared not to be relevant to pyrethroid toxicity in rainbow trout. Pyrethroid interaction with the GABA, receptor was investigated using [³⁵S]TBPS as a radioligand probe and by measurement of GABA-stimulated ³⁶c1- influx in vesicle preparations. At micromolar concentrations, deltamethrin, cypermethrin isomers and other pyrethroids inhibited [³⁵S]TBPS binding by 55- 95% with limited stereoselectivity. Pyrethroids were found to effect a GABAdependent inhibition of [³⁵S]TBPS binding. Ro5-4864, which showed micromolar affinity for the trout PTBR, produced a GABA-modulated interaction with [³⁵S]TBPS binding. These results delineate the reciprocal allosteric interactions between a pyrethroid binding site, a Ro5-4864 binding site, the GABA recognition moiety and the TBPS binding site in trout brain. However, pyrethroids exhibited a modest affinity for this binding site on the GABAA receptor. Pyrethroids indirectly inhibited the GABA-dependent influx of ³⁶Cl⁻into trout brain synaptoneurosomes by increasing the basal uptake of chloride, thereby compromising the ability of the vesicles to respond to applications of GABA. This pyrethroid effect was of nanomolar potency, stereospecific, tetrodotoxinsensitive and mimicked by veratridine. These results suggest that the primary effect of pyrethroids in trout brain, as measured by this assay, was due to an interaction with voltage-dependent sodium channels, increasing sodium conductance and thereby increasing the basal uptake of ³⁶Cl⁻ through a voltagesensitive channel. The convulsant activity of deltamethrin was tested in rainbow trout. The EC₅₀ for convulsant severity was 32 μg /kg body weight. By comparison, pyrethroids at these concentrations in rodents produce no overt toxicity but act as potent proconvulsants. / Graduation date: 1990

The survival and physiology of rainbow trout in alkaline waters was the focus of this thesis. It is known that salmonids have problems with ammonia excretion in alkaline water (Cameron and Heisler, 1983; Wright and Wood, 1985). The first set of studies attempted to increase the survival rates of rainbow trout planted into alkaline lakes by acclimating them to the alkaline conditions before their release. The first field study acclimated rainbow trout to alkaline waters by acidifying the lake water with C0₂ in order to reduce the magnitude of the pH change experienced by the fish. The second field study acclimated rainbow trout to alkaline waters by increasing the alkalinity of the hatchery water in which the fish were held, over a six day period. In both studies the acclimated fish experienced higher survival rates relative to non-acclimated fish. Plasma sodium concentrations ([Na⁺]p1) of the fish were shown to increase, while plasma chloride concentrations decreased. These changes were attributed to an increase in the exchange of external Na⁺ with endogenous H⁺, and the decrease in the exchange of endogenous HC0₃⁻ with external Cl⁻, respectively. The increased [Na⁺]pl may have also been the result of the exchange of plasma ammonium (NH₄⁺) with external Na⁺. The second set of studies investigated the physiological response of rainbow trout to alkaline waters. The first study, the chronic exposure of rainbow trout to alkaline water, showed that trout in hard alkaline water experienced higher survival rates and regulated plasma ammonia and ion concentrations more competently than trout in soft alkaline water. This increased ability to regulate plasma ammonia and ion concentrations was attributed to the possible "reactivation" of the Na⁺/NH₄⁺ exchange. The purpose of the second study, the acute exposure of rainbow trout to alkaline water, was to further investigate the mechanisms that enable fish in hard alkaline water to survive better than fish in soft alkaline water. The possible activity of the Na⁺/NH₄⁺ exchange was again observed in the hard alkaline water. The addition of amiloride to the alkaline hard treatment water increased plasma total ammonia and stabilized [Na⁺]pl′, which supported the "reactivation" of the Na⁺/NH₄⁺ exchange in hard alkaline water. As a result of these studies, the acclimation of rainbow trout to hard alkaline water before being planted into any alkaline body of water was recommended. / Land and Food Systems, Faculty of / Graduate

The effect of transferring fish from water at pH 7.0 to either more acid or more alkaline conditions was to reduce the maximum critical velocity of the fish. In water of pH 4.0, 5.0, and 10.0, the maximum critical velocity was only 54.5%, 66.5%, and 61% respectively of that recorded for fish in the water of pH 7.0. Thus, both acid and alkaline conditions in the water reduce the aerobic swimming capacity of trout. Exposure to acid conditions increased mucus secretion and this was associated with an increase in coughing and breathing frequency in resting fish. Coughing rate increased from 41/hr to 592/hr; and respiration frequency increased from 81/min to 104/min when fish were transferred from water at pH 7.0 to water at pH 4.0. In comparing fish exposed to acid and alkaline waters, the results indicates that fish have a greater capacity to regulate blood pH in acid than in alkaline conditions. The gill potential was strongly dependent on water pH, being negative in neutral water, but positive in acid water and more negative in alkaline solution. Catecholamine levels increased significantly during acid exposure, but were not altered during alkaline exposure. The increasing catecholamine levels appeared at different time periods in different fish during acid exposure and seemed to be associated with the death of the fish. Na⁺ and C1⁻ ion concentrations in plasma decreased significantly after 24hrs of acid exposure, but did not change significantly in alkaline water. This may indicate that ionoregulatory disturbance in plasma is one of the reasons for the decrease in the maximum critical velocity in acid water, but not in alkaline water. / Science, Faculty of / Zoology, Department of / Graduate

Steelhead and rainbow trout are common names for marine-migratory (anadromous) and freshwater-resident forms of Oncorhynchus mykiss, a partially migratory salmonid fish. Anadromous and resident forms are sympatric and can produce offspring with a life history different from their own (i.e., steelhead parents can produce rainbow trout offspring and vice versa). The expression of these alternative life histories is a plastic response to individual patterns of energy acquisition, assimilation, and allocation during juvenile life stages. Individual performance during early stream life is of particular interest because of potential carry-over effects on subsequent growth and developmental trajectories. In a series of experiments in laboratory streams, I determined the influence of individual variation in energy metabolism on behavior, growth, and life-history expression in O. mykiss. Individual variation in energy metabolism was a strong predictor of feeding territory acquisition by juvenile fish during the transition from dependence on maternal provisioning of egg yolk reserves to independent feeding. Feeding territory acquisition was positively associated with standard metabolic rate (SMR) under conditions of an abundant and predictable food supply. When the density of intraspecific competitors was manipulated, the association between SMR and territory acquisition was strongest at intermediate stocking densities, moderate at the highest stocking densities, and weakest at the lowest stocking densities. However, reducing the spatial predictability of food resources within streams reversed the influence of SMR on competitive outcomes. These experiments determined that variation in ecological conditions during early life stages imposes different selection regimes on juvenile O. mykiss and results in physiological divergence among cohorts. Subsequent rearing experiments determined that behavioral dominance influences rates of anadromy and freshwater maturation, most likely through the association between SMR and territory acquisition. In addition to the effects of behavioral dominance, I observed a significant influence of sex, rearing temperature, and individual growth trajectories on the expression of anadromy and freshwater maturation. Partially migratory populations of O. mykiss maintain an exceptionally diverse portfolio of life-history strategies. Results from this work lend insight into a suite of behavioral and physiological processes influencing individual life histories. / Graduation date: 2013