Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 139-147). Also available via the World Wide Web.

The neurohypophysial hormones of Pacific chinook salmon (Oncorhynchus tschawytscha) were purified and the amino acid sequence of both hormones determined. The extraction and purification procedure was developed in an effort to maximize the yields of pure hormones. Pituitary glands were extracted at 4°C using 0.2 M acetic acid. Purification consisted of gel filtration, ultrafiltration, and ion exchange. Gel filtration on Sephadex G-15 columns was used to separate neurohypophysial peptides from high molecular weight material. Separation of the two hormones was accomplished on one of three cation exchangers: carboxymethylcellulose, phosphocellulose or sulfoethyl-Sephadex. The hormones were eluted from cation exchange columns using a sodium or ammonium ion concentration gradient at constant pH; pH 5 was used for chromatography on carboxymethylcellulose and on phosphocellulose; pH 2.45 was used for chromatography on sulfoethyl-Sephadex. The two hormones were purified further by rechromatography of individual hormones on another cation exchange medium. The material obtained by rechromatography was pure as determined by amino acid analyses. The yields of pure hormones at the end of purification procedure was 48% of the starting material. The specific activities of the two purified hormones were 145 and 229 oxytocic units per milligram for Hormone I and Hormone II respectively. The amino acid sequence of Hormone I was determined by a combination of three methods: subtractive Edman degradation, partial acid hydrolysis, and Dansyl-Edman technique. The amino acid sequence of Hormone I was found to be that of 4-serine, 8-isoleucine oxytocin. The amino acid sequence of Hormone II was determined by the Edman subtractive method, the Dansyl-Edman technique, and the mobility of the C-terminal residue on high voltage electrophoresis. The amino acid sequence of Hormone II was found to be that of 8-arginine oxytocin. The two neurohypophysial hormones described from salmon have amino acid sequences identical with those described from four species of Gadiformes and one species of Cypriniformes by other workers. The position of Salmoniformes on the evolutionary tree of teleost fishes suggests that these structures are characteristic of a wide range of teleosts. / Science, Faculty of / Zoology, Department of / Graduate

Two experiments were conducted to study the effect of protein source and level in the diet of juvenile chinook salmon (Oncorhynchus tshawytscha) reared in fresh water tanks. The protein sources compared were a freeze-dried pollock muscle and euphausid mix (9:1)(FPE), three whole herring meals processed differently from the same lot of raw herring and a casein-gelatin mix supplemented with arginine and DL-methionine (CS). The protein sources were tested at three levels of dietary protein in isocaloric diets fed to satiation to duplicate groups of fish for a 42-day period. Protein was replaced by dextrin and glucose on an estimated metabolizable energy basis. The various methods employed to evaluate protein quality yielded different values relative to FPE. In terms of growth rate and assays based on body protein gain, FPE was found to be the best protein source. Low temperature (75°C) drying of herring meal caused a slight reduction in protein quality compared to freeze-drying . High temperature (150°C) dried herring meal was found to be an extremely poor quality protein source. Although high estimates of protein quality were obtained for CS, lower food intake depressed growth in fish fed CS diets. The determination of the endogenous loss of nitrogen from fish enabled the partitioning of protein intake into the amounts used for growth, maintenance and exogenous excretion for each protein source. In Experiment 2, two series of isocaloric diets were tested containing 17 to 47% protein, in increments of 10%, provided by FPE at two levels of dietary energy. The equation y = -0.50699 + 0.25398x - 8.37872x² ,(where y = specific growth rate, and x = protein energy:total energy (PE:TE)) was derived to quantify the dietary protein requirement for juvenile chinook salmon over a 105-day period. Maximum growth was achieved at a PE:TE ratio of 0.55. However, for practical purposes the PE:TE ratio required was found to lie in the range between 0.35 and 0.55. The range permits the fish culturist to consider economic efficiency in diet formulation. / Land and Food Systems, Faculty of / Graduate

Some properties of the adenyl cyclase activity in the maturing testis of Oncorhynchus tshawytscha (chinook salmon) were characterized. The enzymic reaction was linear at 30° and at 15° for at least 60 min. The divalent cation requirement of the salmon testis enzyme was reexamined (33). The optimal concentration of Mg was about 10 mM and of Mn2+ was 5 mM; Mn2+ concentrations above 15 mM caused a marked decrease in enzyme activity. A higher maximal activity was achieved in the presence of Mn2+ than in the presence of Mg2+. Stimulation of the enzyme with the optimal concentration of F-, 12 mM, resulted in a 7-fold increase in the reaction rate over the basal activity. In efforts to solubilize the enzyme, it was found that Lubrol PX and Triton X-100 destroyed enzymic activity but Nonidet P40 and Tween 80 did not. The adenyl cyclase activity in salmon testis homog-enates was stable for at least 6 hours at 0° to 4° but was very unstable at 24°; storage of the homogenate for 24 hours at either 0° to 4° or 24° resulted in a total loss of activity. Differential centrifugation of salmon testis homog-enates which were prepared in isotonic medium revealed tnat all subcellular fractions contained some adenyl cyclase activity. About 55% of the activity sedimented at 600g while only 10% of the activity was recovered in the 105,000g supernatant. The 6300g sediment had a very high specific activity compared with the specific activity of the other fractions. The ATP analogue, adenylyl imidodipho3phate (AMP-PNP), tritium labeled in adenosine, was synthesized from tri-butylammonium imidodiphosphate and adenosine-5’ phosphor-imidazolate. Salmon testis adenyl cyclase catalyzed the conversion of AMP-PNP to cyclic AMP. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

From 1998-2000, laboratory studies were conducted to examine factors that impact saltwater-entry behavior and saltwater preference (SWP) of juvenile chinook salmon, Oncorhynchus tshawytscha. These factors included bacterial kidney disease, stress and the presence of trout, O. mykiss. An additional study investigated the orientation of the startle response of chinook salmon within a salinity gradient. All experiments were conducted in 757-1 tanks in which a stable, vertical salinity gradient was established. SWP was decreased in fish suffering from bacterial kidney disease (31 �� 20.0%), compared with control fish (85 �� 17.6%). A mild chasing stressor resulted in a 26% decrease in SWP relative to unstressed fish. After a severe handling stressor, only 20% of fish preferred salt water, compared with 100% of unstressed controls. After exposure to an overhead predator model, severely stressed fish descended into the saltwater layer, but this response was transient. The presence of non-aggressive steelhead trout did not affect SWP of chinook salmon. Chinook salmon stocked with rainbow trout displayed decreased SWP. Aggression levels in tanks with rainbow trout were higher than in tanks with only chinook salmon. The orientation of the startle response was affected by the presence of salt water. Fish that preferred salt water within a gradient responded by moving horizontally within the saltwater layer. In contrast, control fish (held only in freshwater) moved vertically within the water colunm when startled. Prior preference for salt water superseded the inclination to move upward in the water column when startled. Smoltification involves physiological, behavioral and morphological changes that prepare healthy chinook salmon for seawater residence. However, disease, stress and aggressive interactions can decrease the SWP of fish at this life history stage. Avoidance of salt water during estuarine outmigration is likely maladaptive, and may have ecological ramifications including increased risk of avian predation during outmigration and decreased fitness in the marine environment. / Graduation date: 2002

Environmental factors such as chemical contamination can have immunomodulatory effects on the immune response of fish and may be contributing to the decline in salmonid populations by augmenting disease susceptibility. Xenobiotics can interfere with the immune system at several levels of complexity, and different immune cells and processes have variable sensitivity to pollutants. For this reason, a suite of tests is required to evaluate immunomodulatory mechanisms. In this thesis, I formulated and calibrated an assay for the detection of humoral immunity for chinook salmon (Oncorhynchus tshawvtscha). Subsequently, I used this technique in conjunction with other immune and endocrine assays to detect effects of embryonic exposure to o,p'-DDE, a known environmental estrogen. The technique combines exposure of whole animals or leukocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leukocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccaride (LPS) was quantified by flow cytometric analysis of forward and side scatter properties. In addition, I used a fluorescein isothiocyanate labeled anti-rainbow trout surface immunoglobin monoclonal antibody (anti-RBT SIgM-FITC) to determine the ability of the lymphoblasts to express surface immunoglobin (SIgM) through flow cytometry. I used the assay to evaluate the effects of short-term exposures to o,p'-DDE during early life history stages on the long-term immune competence of fall chinook salmon. Immersion of chinook salmon eggs in 10 ppm o,p'-DDE for 1 h at fertilization followed by 2 h at hatch caused significant reductions in the ability of splenic leukocytes to undergo blastogenesis and express SIgM upon in vitro stimulation with LPS one year after treatment (ANOVA, P<0.05). The concentration of o,p'-DDE in fry treated with 10 ppm o,p'-DDE was 0.92 ��g g����� lipid one month post first feeding. The chemical persisted through development and, one year after exposure, levels in juvenile muscle tissue were 0.94 ��g g����� lipid. Mortality rate, time to hatch, fish size, sex ratios, gonadal development, plasma estradiol and 11-ketotestosterone concentrations were not affected by treatment with o,p'-DDE. In addition, neither plasma lysozyme concentration, nor mitogenic response of splenic leukocytes to concanavallin A or polyinosinic-polycytidylic acid were influenced by the treatment. A short period of exposure to an estrogenic chemical during early periods of development induced long term effects on humoral immune competence of chinook salmon. I discuss the possibility that the xenobiotic is exerting its activity through steroid-mediated pathways. / Graduation date: 2002

The spawning habitat associated with fall chinook salmon (Oncorhynchus tshawytscha) redd clusters was investigated in the Hanford Reach of the Columbia River. A conceptual spawning habitat model is proposed that describes how geomorphic features of river channels create hydraulic processes, including hyporheic flows, that influence where salmon spawn in unconstrained reaches of large mainstem alluvial rivers. Spatial point pattern analysis of redds showed that redd clusters averaged approximately 10 hectares in area and their locations were consistent from year to year. The tendency to spawn in clusters suggests fall chinook salmon's use of spawning habitat is highly selective. Hydraulic characteristics of the redd clusters were significantly different than the habitat surrounding them. Velocity and lateral slope of the river bottom were the most important habitat variables in predicting redd site selection. While these variables explained a large proportion of the variance in redd site selection (86 to 96%), some unmeasured factors still accounted for a small percentage of actual spawning site selection. Further investigation showed that the magnitude and chemical characteristics of hyporheic discharge were different between and within two spawning areas. Apparently, fall chinook salmon used chemical and physical cues from the discharge to locate spawning areas. Traditional spawning habitat models could be improved if they: used spawning area-specific, rather than river-specific; spawning characteristics; incorporated hyporheic discharge measurements; and gave further consideration to the geomorphic features that are present in the unconstrained segments of large alluvial rivers. Ultimately the recovery of endangered fall chinook salmon will depend on how well we are able to recreate the characteristics once common in alluvial floodplains of large rivers. The results from this research can be used to better define the relationship between these physical habitat characteristics and fall chinook salmon spawning site selection, and provide more efficient use of limited recovery resources. / Graduation date: 1999

Three experiments were designed to determine if and how steelhead trout, Oncorhynchus mykiss, may affect chinook salmon, O. tshawytscha, when they are confined together as in a raceway or on a barge. We observed groups of chinook and steelhead together and groups of only chinook in a behavioral experiment to determine if steelhead are aggressive and if their presence changed the behavior of chinook. Two physiological experiments were completed to determine if the loading of steelhead on top of chinook and if the introduction of odor from rainbow trout (steelhead not available) caused a change in plasma cortisol levels in chinook. It was found that chinook showed characteristics of a schooling species, while steelhead exhibited territory holding characteristics. Behavioral changes in chinook were observed when steelhead were present. Chinook grouped with steelhead reduced their movements, darted less, were attacked up to 16 times more often, and were found less frequently in the shade than groups of only chinook. Steelhead were found to establish territories and defend them with chases, charges, and nips. In attempts to establish territories and defend them, steelhead attacked chinook as often as they attacked other steelhead even though chinook showed little aggression toward steelhead. In a physiological experiment, chinook experienced the loading of salmonids into their tank. Chinook had higher levels of plasma cortisol at 2 and 32 hours after the loading of steelhead than chinook that were loaded with chinook or controls (no loading). A second physiological experiment with odor showed that chinook that received rainbow odor and those that received chinook odor had similar levels of plasma cortisol. Cortisol levels (two hours after the odor was introduced) were higher in chinook receiving either of the scented waters than in those that did not receive any odor. In conclusion, all three experiments indicated that the presence of juvenile steelhead trout affect juvenile chinook salmon behavior and physiology. / Graduation date: 1998

Graduation date: 1996

I examined habitat electivity and movement patterns of adult spring chinook salmon at microhabitat and channel unit spatial scales, and seasonal to annual temporal scales in seven streams in the Grande Ronde, John Day, and Imnaha basins. The objective was to compare habitat use and availability among streams, channel units, and microhabitats, and to assess chinook salmon fidelity to those habitats using radio-telemetry. The analyses showed that habitat quality and availability in the seven study streams varied. Each stream posed different physical constraints on adult chinook salmon habitat; this was reflected by the differential use of habitat by salmon among streams. Salmon elected pools almost exclusively in the John Day Basin, whereas pools and riffles were elected in near equal proportion in the Grande Ronde and Inmaha basins. Within streams, use was similar between years. Almost all salmon were observed in association with cover, but the type of cover largely reflected availablity. Chinook salmon elected the deepest depths within channel units (microhabitat scale), but not necessarily the deepest channel units among streams (channel unit scale). Chinook salmon did not elect cooler stream temperatures within channel units in any study stream, except the Middle Fork John Day River. Radio-tagged chinook showed a high fidelity to habitats, except when stream temperatures approached lethal limits. Due to stream specific differences in habitat availability and use, multiscale habitat assessments for individual streams are recommended to increase the success of watershed restoration activities. / Graduation date: 1999