Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 79-93).

Growth hormone-releasing hormone (GHRH) is a neuropeptide which stimulates the synthesis and release of growth hormone (GH) from the pituitary gland. The primary structure of this peptide has been identified in 7 mammalian species while the gene has been isolated from only rat and human. GHRH is a member of the glucagon superfamily which includes vasoactive intestinal peptide (VIP), glucagon, secretin, peptide histidine methionine (PHM), gastric inhibitory peptide (GIP) and a recently identified peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The evolutionary relationships of this superfamily are not well understood because the gene structure of these molecules has only been identified in mammals. This thesis presents immunological evidence of a GHRH-like molecule, and identifies a GHRH/PACAP precursor and gene that encode two peptides, a GHRH-like molecule structurally related to PACAP-related peptide (PRP) and PACAP, from sockeye salmon brain. An antiserum directed against a topologically assembled epitope of human GHRH 1-44 (NH2) was produced and used to develop a radioimmunoassay for detection of immunoreactive GHRH in brain extracts of salmon, guinea pig, mouse and alligator. An immunoreactive GHRH from salmon brain extracts with a retention time on reverse phase high-performance liquid chromatography (HPLC) distinct from human GHRH was present. In alligator, the same antiserum also detected a GHRH-like molecule. During attempts to purify alligator GHRH, alligator brain neuropeptide Y (NPY) was identified. Alligator NPY is the first non-mammalian vertebrate to have 100% sequence identity to human NPY. The sequence identity between alligator and human NPY suggests that this sequence is the same as the ancestral amniote NPY. Molecular biological techniques were used for the structural identification of the salmon GHRH-like molecule and another peptide. The salmon GHRH/PACAP precursor contains 173 amino acids and has dibasic and monobasic processing sites for cleavage of a 45 amino acid GHRH-like peptide with a free acid C-terminus and a 38 amino acid PACAP with an amidated C-terminus. The salmon GHRH-like peptide has 40% amino acid sequence identity with the human GHRH and 56% identity with human PACAP-related peptide (PRP). Salmon PACAP-38 is highly conserved (89%) with only 4 amino acid substitutions compared with the human, ovine and rat PACAP-38 peptides. Nucleotide sequencing and use of the polymerase chain reaction show the exon/intron organization of the salmon GHRH/PACAP gene to be similar to the human PACAP gene. Unlike the mammalian PACAP genes, the salmon gene produces two precursor forms by post-transcriptional processing. One form is similar to the mammalian PACAP precursors, while the second form is shorter due to the excision of exon 4. This deletion results in the loss of the first 32 amino acids of the GHRH-like peptide from the precursor. The high sequence identity and structural organization between the GHRH(PRP)/PACAP and PHM(PHI)/VIP genes suggest a duplication event occurred in an ancestral gene after the divergence from the other glucagon superfamily members. GHRH in mammals may have arisen by gene duplication after the divergence of the tetrapods from the other vertebrate lines. Thus, GH in fish may be controlled by the two molecules, GHRH-like peptide and PACAP, located on a single GHRH/PACAP gene. / Graduate

In the following study, I tried to link hormonal background conditions to successful olfactory imprinting in sockeye salmon by employing behavioural, endocrinological and electrophysio logical experiments. In the initial experiments, sockeye salmon were exposed to potential imprinting odorants, with or without additional treatment with thyroid hormones, during several juvenile stages between fertilization and beyond the PST. After two years of rearing, these fish were tested for behavioural responses to test odorants in two behavioural arenas. Neither immature nor mature fish reacted behaviourally to the odorants that they had been exposed to previously. Therefore, exposure of juveniles to odorants did not lead to imprinting to those odorants under hatchery rearing conditions. In contrast, juvenile fish that were exposed to test odorants and treated with a combination of T₃ and T₄ (in all cases) or T₃ (in one case) the two most common forms of thyroid hormones, did exhibit an odorant recognition response two years later. However, the response differed between immature and mature fish. Mature fish were attracted to the imprinting odorant, whereas immature fish were repelled by the it. When immature fish were injected with GnRH before testing, their behavioural response was reversed. No behavioural response could be detected in fish that had been challenged with either T₃ or T₄ alone, in contrast to a combined treatment with both forms. Thus, I found evidence that a combination of T₃ and T₄ initiated imprinting and that GnRH motivated odorant recognition. To examine the underlying hormonal processes, I first determined plasma thyroid hormone concentrations in sockeye salmon before and after hormonal challenges with thyroid hormones or GnRH. In addition, the activity of the deiodinase enzyme that converts T₄ into the other possible forms of thyroid hormones was investigated in sensory and non-sensory tissues. The results suggested that only a combined T₃T₄ treatment increased the availability of both thyroid hormone forms in blood plasma, while a separate challenge with T₄ suppressed T₃ availability and vice versa. Moreover, the results provided evidence for deiodinase activity in the olfactory epithelium and the retina and demonstrated that GnRH can modulate the T₄ conversion process. This inform ation was helpful for planning and interpretation of the remaining experiments. Results obtained from a classical conditioning paradigm (heart-rate-conditioning), provided support for the hypothesis that GnRH lowers the threshold to an imprinting odorant and that the influence of GnRH was not restricted to an enhancement of motivation. To investigate whether hormonal action could also modulate the sensitivity of the peripheral olfactory system, electrophysiological responses from the olfactory epithelium (electro-olfacto-grams or EOGs) were recorded. The EOG results established that thyroid hormones and GnRH increased the EOG response of adult naïve (never imprinted to an odorant) fish, as did maturity. In the last experiments, I conducted EOG recordings on fish that had been imprinted at a juvenile stage. In summary, EOG recordings revealed that the imprinting process increased sensitivity to the imprinting odorant at maturity, while sensitivity in immature fish was decreased in comparison to non-imprinted fish. In combination with my behavioural results, this could explain why salmon do not enter their natal stream before they reach maturity. At maturity however, I also encountered desensitization to non-imprinting odorants, which might increase the ability to focus the olfactory system to the task of homing. / Graduate

This study was aimed at providing information that could be used to estimate the predation effect of squawfish on sockeye salmon. Initial studies at Griffen Lake revealed that the percentage of the population acting as predators, and the average stomach volume, increased with increasing size in non-spawning squawfish. Squawfish in Griffen Lake appeared to be most active at night. For the most part, a force fed volume (2.0 ± .1 gms) was digested by all sizes of squawfish in less than 24 hours. Further studies at Cultus Lake in the laboratory revealed that the rate of digestion was dependent on temperature, volume of food, and size of fish. At 6°C a lag prior to digestion commencing was observed and digestion was not complete for the medium volume in less than 52 hours. On an average the medium fed volume was digested in less than 24 hours for temperatures greater than 15°C. Medium volume corresponded to approximately twice the smallest volumes recorded in stomach contents in the field. A life span of ten years for males and 14 years for females with no differential in the growth rate was determined from the banding patterns and sections of pharyngeal teeth. Consumption rates and periodicity of feeding and activity were within the limits of data from Griffen Lake. Laboratory calculated routine metabolic rate was approximately twice the theoretical rate for pooled species. A tagging experiment at Cultus Lake revealed a population of approximately 20, 000 squawfish greater than 720 cm that, on an average, grew less than 0.36 cm during the winter. Growth during the summer was assessed to be in the form of weight increase of body tissue and gametes. Trap catches and temperature preference experiments indicated that squawfish are found within particular temperature regimes in different phases of the year. Within Cultus Lake, distance did not prevent squawfish aggregation on concentrations of sockeye smolts. There was increased consumption of smolts during smolt migration from the lake. In the field a significant difference in rate of digestion for different sizes of squawfish could not be demonstrated; however, there was a difference in the volume voluntarily consumed. With these findings and other theoretical information, it is possible to determine quantitatively the predation effect of a squawfish population on sockeye. Having established a population estimate, an estimate of annual mortality (55.2 per cent per year), and the temperature specific phases (early smolt phase, peak smolt phase, spawning phase, summer phase, fall phase, winter phase), two methods for the assessment of the predation effect were possible. First, knowing proportion of the size classes acting as predators, the numbers and frequency distribution of squawfish remaining within any phase, the duration and temperature characteristics of the phase, the effect of temperature on the rate of digestion, and the volumes that can be digested in 24 hours, it was possible to get an accumulated volume consumed for the population. For the 20, 000 squawfish greater than 20 cm fork length in Cultus Lake, this represents an approximate consumption of 1.4 million sockeye smolt equivalents. The second estimate of consumption was based on the energy requirements converted to consumption rates using conversion coefficients for the same population. The energy requirements to complete spawning, growth and mean metabolism were summed, then converted to a consumption volume for the population. The findings revealed that approximately 2.8 million sockeye equivalents are required. / Science, Faculty of / Zoology, Department of / Graduate

Data from five conductivity-temperature-depth (CTD) surveys collected during 1985 and 1986 in support of project MOIST -Meteorological and Oceanographic Influences on Sockeye Tracks- are used to describe the salient oceanographic features of the waters lying between Vancouver Island and the British Columbia mainland coast. Using these data, four oceanographic regimes are clearly defined on the basis of salinity structure. Temperature-Salinity diagrams are used to discuss water types and mixing ratios in these regimes. Vigorous tidal mixing over shallow sills and/or in narrow channels produces tidally mixed fronts that separate oceanographic regimes. The tidal evolution of two of these fronts located near Weynton Passage and Cape Mudge are discussed by means of 24-hour CTD stations. Seasonal variability of the residual estuarine circulation is examined and estimates of the seaward flow in the upper layer of a very simple two-layer geostrophic model were found to be in reasonable agreement with the few direct measurements made in this region. Seasonal variability of the general hydrography is described. Ultrasonic telemetry provided horizontal and vertical distribution time series data for return migrating sockeye salmon (Oncorhynchus nerka). Concurrent high spatial resolution CTD data was used to specify the ambient temperature and salinity fields in the immediate vicinity of the tagged sockeye. Spectral analysis of the depth and ambient oceanographic data time series revealed periodic vertical movements at approximately 15 and 33 minutes per cycle for fish tracked in the slightly stratified regimes of Queen Charlotte Strait, western Johnstone Strait and the Strait of Georgia. High frequency large amplitude periodic vertical movements were characteristic of fish that did not make significant progress towards the Fraser River: low frequency small amplitude vertical movements were characteristic of well oriented fish. Aspect ratio, defined as the horizontal distance travelled divided by the vertical distance travelled, gave an indication of the relative degree of homeward orientation. Vertical distribution and orientation were also related to the frequency and duration of successive vertical excursions. Fish depth and vertical swimming velocity were found to be positively correlated in regions of weak stratification and/or for well oriented fish. Ambient density gradients were not found to inhibit vertical movements as the rate of doing work against hydrodynamic drag was several orders of magnitude greater than that of doing work against a varying buoyancy force. In the presence of strong temperature and salinity gradients, tracked sockeye were most often observed at depths not associated with the maximum gradients. In stratification regimes where temperature and salinity gradients were nearly uniform with depth, tracked sockeye were observed at depths uniformally distributed throughout the thermo- and haloclines. Minimum vertical swimming velocities were generally associated with minimum vertical gradients. These observations suggest that the tracked sockeye frequently swam through but did not reside in the region of the maximum gradients. Dimensional analysis suggested that physical variables alone are insufficient to specify the vertical distribution of the tracked sockeye. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

Laboratory experiments were conducted to study the effects of dehydro-abietic acid (DHA) on the physiology of the adaptation of sockeye salmon smolts (Oncorhynchus nerka) to sea water. Dehydroabietic acid occurs in the rosin of commercially important coniferous trees and is found in the untreated effluents of the pulp and paper industry at concentrations acutely toxic to salmonids. As this resin acid is known to be one of the more persistent toxic components of kraft mill effluent (KME) and although its concentrations are greatly reduced by biological treatment, DHA is nevertheless discharged in the effluents of the pulp mills situated on the Fraser River system as well as of those located on the coast of British Columbia. As sockeye salmon utilize both the Fraser and Thompson Rivers during their downstream migration, this species may be exposed to DHA before entering the sea. An attempt was made to simulate this situation in the laboratory by exposing sockeye salmon smolts to a sublethal concentration of DHA (0.65 mg/L) in fresh water for 120 h and then transferring them into sea water (28 °/oo) containing no DHA. Hydromineral balance was studied by monitoring changes in plasma osmolality, plasma NA⁺, K⁺ , Ca⁺⁺, Mg⁺⁺ and Cl⁻, blood hematocrit and muscle water content at the end of the freshwater DHA exposure and at 24 h intervals during the adaptation to sea water (120 h). After 24 h in sea water the gill permeability to water and the water transport ability of the gut were also determined. Supportive experiments measured changes in the size of red blood cells, the levels of plasma bilirubin as well as the uptake and tissue distribution of DHA in sockeye salmon smolts. Lipid extracts of various tissues were analyzed for DHA residues by gas chromatography coupled with mass spectrometry (GC-MS). The exposure of sockeye salmon to DHA in fresh water resulted in a hydromineral disturbance characterized by a drop in plasma osmolality, sodium, and chloride, indicating a general hydration which was reflected by increased muscle water content. A lowering of dissolved oxygen to 75% saturation markedly increased the toxicity of DHA and the osmotic imbalance may have been a secondary result of an adaptive respiratory response to a hypoxic stress brought on by DHA exposure. Increases in blood hematocrit were caused by a swelling of the red blood cells related to lowered plasma osmolality. When these fish were transferred to sea water, the hydration was replaced by dehydration and a rise in osmolality was caused by abnormally elevated levels of all the plasma ions. The added salinity stress resulted in some mortality and considerably greater excursions in plasma electrolytes occurred in fish which were experiencing locomotor difficulty. Plasma magnesium showed the greatest elevation and took the longest (96 h) to return to normal levels. Prior DHA exposure increased the permeability of the gill. During acute DHA exposure in fresh water a gradual deterioration in schooling and fright response was followed by hypersensitivity to mechanical stimuli and abnormal swimming behavior. After sublethal exposure, the reduction in schooling and fright response generally became most evident during the first 24 h of sea water adaptation. These results of the study are discussed in terms of the possible roles played by the gills, gut and kidney in the DHA-induced perturbations of hydromineral balance. The implications of the accompanying alterations in behavior are discussed in the context of the ecological survival of sockeye salmon smolts during adaptation to sea water. Residue analyses showed that sockeye salmon accumulated DHA from the water to high levels in the brain (954 x), liver (428 x) and kidney (404 x) as well as in other tissues. The presence of DHA metabolites in the bile, which also contained the highest DHA residues (647.3 μg/g), indicates that the hepatobiliary route is important in the excretion of DHA by fish. The possibility of the bioaccumulation of DHA by fish in the wild is discussed in relation to the setting of water quality criteria for pulp mill effluent. / Science, Faculty of / Zoology, Department of / Graduate

The predatory functional response is one of the important components of the interaction between predator and prey populations. This response has not been measured for fish predators and fish prey, in spite of the demonstrated importance of predation in regulating numbers in populations such as the juvenile salmon system used in this thesis. Laboratory exeriments were conducted to determine the form of the functional response of the prickly sculpin (Cottus asper), a common freshwater predator, to density of sockeye salmon fry, Oncorhynchus nerka. An alternative prey, chum salmon eggs (Oncorhynchus keta), were presented to the predator with the salmon fry, in order to facilitate switching at low fry densities and possibly lead to a sigmoid (type III) response. Initial experiments showed that sculpin feeding rate did not substantially differ from day to night; that in the absence of food, hunger increased to a maximum after about seven days' starvation; and that a density of 400 chum salmon eggs in a 2000 1 tank produced maximum consumption level by sculpins of 160 mm total length. The functional response experiments did not indicate a sigmoid rise in consumption over low fry densities. However, the initial decelerated rise in consumption was followed by an anomalous drop in response and a subsequent rise in response. It is suggested that this may result from the summation of separate responses through different sensory modalities, or from interference with predation by fry at intermediate densities. This shape of functional response may imply a stable local equilibrium at intermediate densities of salmon. Sculpins probably have a significant impact on fry numbers only at very localized points in space and time, such as at the outlets of tidal creeks on a falling tide. / Science, Faculty of / Zoology, Department of / Graduate

Prior to the construction of the dams, migrating salmonid species accessed the upper reaches of the Capilano, Seymour, and Coquitlam watersheds as an integral part of their natural lifecycle. There are multiple initiatives being undertaken by BC Hydro and the stakeholders to re-introduce sockeye salmon (Oncorhynchus nerka) to one of their natural habitat in the upper Coquitlam watershed. Concerns have been raised that the restoration may be in conflict with the drinking water mandate of Metro Vancouver’s watershed management policies. The research objective of this project was to investigate and assess the social, economic, and environmental aspects of restoring an extirpated sockeye salmon population. Through an exploratory case-study approach, the research concluded that the existing fish passage constraints and reliance upon re-anadromy to restore the population are limiting factors in sustaining the Coquitlam Reservoir sockeye.

Thesis (Ph. D.)--University of Washington, 1989. / Vita. Includes bibliographical references (leaves [194]-207).

The locomotor responses of juvenile and adult sockeye salmon (Oncorhynchus nerka) to concurrent changes in temperature and salinity were examined in a controlled laboratory setting. I hoped to better understand how these environmental factors influence the coastal movements of migrating salmon. Juvenile sockeye were captured during the downstream migration from Great Central Lake on Vancouver Island, British Columbia, Canada. The fish were acclimated for 1 wk at 10°C, 20 ppt, and then tested in annular activity tanks. Spontaneous locomotor movements were recorded during concomitant changes in temperature and salinity using infra-red photometry. Raising the water temperature by 4°C in 1 h caused a dramatic increase in locomotor activity. Decreasing temperature by 4°C or varying salinity by 10 ppt from the control levels did not influence routine swimming speed and there was no interaction between factors. Adult sockeye homing to the Fraser River, British Columbia, Canada were captured along the nearshore migration route in two oceanographically distinct regions. Three groups of fish were collected from the cold, saline waters of Queen Charlotte Strait, near the northern end of Vancouver Island. Two groups of sockeye were captured within 60 km of the Fraser River in the warmer, less saline waters of the Strait of Georgia. The adults were acclimated 2-5 days at 12°C, 30 ppt and locomotor activity was tested in annular activity tanks. Routine swimming speed and turning rate rose when the water temperature was raised by 4°C in 2 h, however, locomotor activity was not influenced by decreasing temperature. In addition, decreasing salinity by 10 ppt in 2 h had no effect on swimming activity of adult sockeye and there was no interaction between the two factors. Fish taken from the Strait of Georgia generally showed a less dramatic response to increasing temperature than adults captured in Queen Charlotte Strait. Results indicate that warm coastal temperatures may influence the nearshore migration of both juvenile and adult sockeye salmon. / Science, Faculty of / Zoology, Department of / Graduate