The seafood industry is of considerable importance to both the New Zealand and global economies and therefore tissue preservation technologies that increase product quality and/or prolong shelf life have the potential to add significant value. Technologies for maintaining the viability of isolated tissues also have a wide range of other medical and industrial applications. This thesis examines the relationship between metabolic function, oxidation and cell death and the resulting stability of the non-viable tissues during long term storage in chinook salmon (Oncorhynchus tshawytscha) red and white skeletal muscle tissue. This research also looks at the role of the aquatic anaesthetic AQUI-S™, in which the active ingredient is isoeugenol (a lipid soluble antioxidant), and other antioxidant compounds in preserving metabolic function in viable tissues and tissue stability in nonviable tissues. Perfusion of salmon tails at 15℃ over 5 or 10 hours with oxygen saturated saline resulted in significant increases in protein and lipid oxidation (protein carbonyl and TBARS concentrations respectively) in the red muscle, but not the white muscle. The introduction of ascorbic acid and uric acid into the saline did not reduce the oxidation in the red muscle despite significantly increasing their respective concentrations in the tissue. This indicates the difficulties associated with attempting to extend tissue viability by delivering free oxygen to the tissue and also highlights the difference in susceptibility of the two muscle types to oxidation. Tail fillets from salmon harvested in both rested and exhausted physiological states using AQUI-S™, and fillets from exhausted salmon harvested without AQUI-S™, were exposed to air at 15℃ for up to 96 hours. Protein carbonyls increased in a roughly linear fashion over the entire 96 hours in all three groups. Both lipid peroxides (TBARS) and uric acid concentrations began to increase in the exhausted group after 30 hours. In contrast, no significant increases in lipid peroxides or uric acid was seen in the fillets from either group harvested using AQUI-S™. Vitamin E concentrations reduced slowly but did not change significantly despite the oxidation that was evident in the tissue. These processes also occurred in salmon tail fillets during storage at 6℃. The measurement of ATP related compounds provides an effective indicator of both the metabolic state of the tissue post-harvest and the quality. The breakdown of these compounds is also associated with the production of ammonia and hydrogen peroxide. Fresh rested salmon fillets had high concentrations of ATP and creatine phosphate, which were both depleted after 12 hours storage at 15℃. This indicates that cell viability lasted a number of hours following harvesting. These metabolites were depleted in exhausted fillets and metabolic potential appeared to be immediately compromised. The concentration of the taste enhancing compound IMP was significantly reduced in fresh rested tissue, but increased during storage, and was significantly higher than in exhausted tissues following 12 hours of storage at 15℃. This indicates that some properties of rested tissues may improve with limited storage times. The accumulation of uric acid - the metabolic end point for ATP related compounds - was also significantly reduced in rested tissue and increases in K-value were slowed. AQUI-S™ showed an ability to preserve tissue function through its anaesthetic action allowing tissue to be harvested in a rested state, and to reduce late stage lipid oxidation in stored salmon tail fillets. The antioxidant action of isoeugenol in salmon fillets may be mediated through its ability to chelate transition metals released during tissue degradation. This research shows that during reperfusion and during fillet storage there is a significant level of oxidative stress, which needs to be minimized while maintaining basic tissue metabolism to prolong tissue and cellular viability. The development of future technologies to preserve tissue viability may depend on the development of a synthetic oxygen carrying compound with properties similar to red blood cells. This may allow more control over oxygen delivery, potentially reducing the oxidative stress associated with high concentrations of free oxygen in solution. However, preserving cell viability will also require the maintenance of endogenous antioxidant function and there is also the potential to use iron chelating compounds including plant derived flavonoids to preserve non-viable tissues. Future research in these areas is necessary.

The seafood industry is of considerable importance to both the New Zealand and global economies and therefore tissue preservation technologies that increase product quality and/or prolong shelf life have the potential to add significant value. Technologies for maintaining the viability of isolated tissues also have a wide range of other medical and industrial applications. This thesis examines the relationship between metabolic function, oxidation and cell death and the resulting stability of the non-viable tissues during long term storage in chinook salmon (Oncorhynchus tshawytscha) red and white skeletal muscle tissue. This research also looks at the role of the aquatic anaesthetic AQUI-S™, in which the active ingredient is isoeugenol (a lipid soluble antioxidant), and other antioxidant compounds in preserving metabolic function in viable tissues and tissue stability in nonviable tissues. Perfusion of salmon tails at 15℃ over 5 or 10 hours with oxygen saturated saline resulted in significant increases in protein and lipid oxidation (protein carbonyl and TBARS concentrations respectively) in the red muscle, but not the white muscle. The introduction of ascorbic acid and uric acid into the saline did not reduce the oxidation in the red muscle despite significantly increasing their respective concentrations in the tissue. This indicates the difficulties associated with attempting to extend tissue viability by delivering free oxygen to the tissue and also highlights the difference in susceptibility of the two muscle types to oxidation. Tail fillets from salmon harvested in both rested and exhausted physiological states using AQUI-S™, and fillets from exhausted salmon harvested without AQUI-S™, were exposed to air at 15℃ for up to 96 hours. Protein carbonyls increased in a roughly linear fashion over the entire 96 hours in all three groups. Both lipid peroxides (TBARS) and uric acid concentrations began to increase in the exhausted group after 30 hours. In contrast, no significant increases in lipid peroxides or uric acid was seen in the fillets from either group harvested using AQUI-S™. Vitamin E concentrations reduced slowly but did not change significantly despite the oxidation that was evident in the tissue. These processes also occurred in salmon tail fillets during storage at 6℃. The measurement of ATP related compounds provides an effective indicator of both the metabolic state of the tissue post-harvest and the quality. The breakdown of these compounds is also associated with the production of ammonia and hydrogen peroxide. Fresh rested salmon fillets had high concentrations of ATP and creatine phosphate, which were both depleted after 12 hours storage at 15℃. This indicates that cell viability lasted a number of hours following harvesting. These metabolites were depleted in exhausted fillets and metabolic potential appeared to be immediately compromised. The concentration of the taste enhancing compound IMP was significantly reduced in fresh rested tissue, but increased during storage, and was significantly higher than in exhausted tissues following 12 hours of storage at 15℃. This indicates that some properties of rested tissues may improve with limited storage times. The accumulation of uric acid - the metabolic end point for ATP related compounds - was also significantly reduced in rested tissue and increases in K-value were slowed. AQUI-S™ showed an ability to preserve tissue function through its anaesthetic action allowing tissue to be harvested in a rested state, and to reduce late stage lipid oxidation in stored salmon tail fillets. The antioxidant action of isoeugenol in salmon fillets may be mediated through its ability to chelate transition metals released during tissue degradation. This research shows that during reperfusion and during fillet storage there is a significant level of oxidative stress, which needs to be minimized while maintaining basic tissue metabolism to prolong tissue and cellular viability. The development of future technologies to preserve tissue viability may depend on the development of a synthetic oxygen carrying compound with properties similar to red blood cells. This may allow more control over oxygen delivery, potentially reducing the oxidative stress associated with high concentrations of free oxygen in solution. However, preserving cell viability will also require the maintenance of endogenous antioxidant function and there is also the potential to use iron chelating compounds including plant derived flavonoids to preserve non-viable tissues. Future research in these areas is necessary.

Salmonids are a diverse group of fishes whose common ancestor experienced an evolutionarily important whole genome duplication (WGD) event approximately 90 MYA. This event has shaped the evolutionary trajectory of salmonids, and may have contributed to a proliferation of the repeated DNA sequences known as transposable elements (TEs). In this work I characterized repeated DNA in five salmonid genomes. I found that over half of the DNA within each of these genomes was derived from repeats, a value which is amongst the highest of all vertebrates. I investigated repeats of the most abundant TE superfamily, Tc1-Mariner, and found that large proliferative bursts of this element occurred shortly after the WGD and continued during salmonid speciation, where they have produced dramatic differences in TE content among extant salmonid lineages. This work provides important resources for future studies of salmonids, and advances the understanding of two important evolutionary forces: TEs and WGDs. / Graduate / 2019-04-19

This dissertation describes a proposed methodology for Cumulative Effects Assessment (CEA) with the purpose of improving the process by making it both more substantive and quantitative. The general principles of the approach include the following: use of effect-based analyses where selected Valued Component (VC) sensitivities are identified first and then effect pathways are determined building bottom-up linkages from VC sensitivities to potential stressors or combinations of stressors to effect drivers and forces behind the drivers. Models were developed based on statistical or historic trend analysis or literature review that predicted the responses of the VCs to changes in effect drivers. Further, scenarios of divergent futures were created that involved different developments of each effect driver or force, and finally the models were applied to each scenario to project the state of the studied VCs. A practical implementation was conducted to demonstrate the use of the proposed methods on future population trends of two anadromous salmon species from the Cowichan River, British Columbia, Chinook and Coho. The assessment was conducted for both early freshwater and marine phases of their life. For the freshwater phase, the assessment focused on two main factors affecting salmon survival, streamflow and stream temperature and established two main drivers affecting these stressors, land use and climate change, and two main forces behind these drivers, Local and Global human development driven change, respectively. Effects of stream temperature and streamflow on salmon freshwater survival were simulated using two models; one was based on Chinook freshwater survival correlations with stream temperature and was developed only for Chinook, and the other was based on literature-derived temperature and streamflow thresholds and was developed for both species. Connections between the stressors (stream temperature and streamflow) and drivers (land use and climate change) were established through a hydrologic model and stream temperature regression model. For the marine environment, models were created using Pearson correlation and stepwise regression analysis examining links between survival of Cowichan River Chinook and Strait of Georgia hatchery-raised and wild Coho and various environmental variables of the nearshore zone of Strait of Georgia and Juan de Fuca Strait. The models were applied to project future salmon survival under four future scenarios for 2050 that were created by combining two opposite scenarios of land use in the watershed, forest conservation and development, and two climate change scenarios, extreme and moderate. Scenario projections showed a decrease in overall (combined early freshwater marine) survival by 2050 for all three studied salmon populations. None of them are likely to survive in scenarios with extreme climate change, while scenarios with moderate climate change showed positive survival rates although lower than present-day baseline levels. Analysis also showed that land use management within the Cowichan River watershed can also affect freshwater survival of both Chinook and Coho and marine survival of Chinook through influence of river discharge on nearshore processes. However, our land-use management scenarios have considerably weaker effect than climate change on salmon survival. Therefore, we conclude that land use management alone is not sufficient to offset effects of climate change on salmon survival. / Graduate

Cumulative effects from multiple anthropogenic stressors over the past three centuries have severely impacted estuarine and coastal habitats, with cascading effects on the species that rely upon them. Pacific salmon (Oncorhynchus sp.) are migratory species that use estuaries as juveniles and as adults and deliver critical nutrients to coastal ecosystems as they move between fresh and marine waters. Many once abundant salmon populations have been extirpated or are in severe decline relative to historic levels, yet the strength of the relationship between habitat loss and population productivity has been challenged. In this dissertation, I applied field studies, otolith analyses, and conservation decision science tools to investigate the relative importance of estuarine habitat to salmon populations, with the aim of advancing effective management solutions for these species and their habitats. First, I conducted a two-year field survey of fish communities in the Fraser River estuary, British Columbia, Canada comparing the species richness and relative catch amongst three distinct habitats. I found that this impacted estuary still supported a rich community of migratory marine and anadromous fishes, as well as resident estuarine fish species. Each habitat supported some unique fish assemblages, with eelgrass supporting the highest catch and diversity of fishes overall but brackish marsh supporting the highest and most consistent catch of salmonids. Next, I used otolith analyses to quantify the residency and growth of juvenile Chinook salmon in the estuary. I found that for one of the only two remaining Chinook salmon stocks abundant enough to still support limited harvest in the Fraser River, the estuary provides vital rearing habitat, with juveniles residing in the estuary for an average of 6 weeks, during which time they had mean daily growth rates of 0.57 mm fork length, approximating growth in healthier estuarine systems. The use of these habitats by juvenile Chinook salmon had not been quantified previously, so these findings directly inform management of this population, which was recently designated as Threatened by the Committee on the Status of Endangered Wildlife in Canada. Finally, I applied Priority Threat Management, a conservation decision science framework, to predict the future status of Pacific salmon in the lower Fraser River and identify the most cost-effective conservation solutions out of a suite of alternative management strategies. On our current trajectory none of these populations were predicted to be assessed as ‘green’ or healthy status at the end of 25 years. In contrast, implementation of broad scale habitat restoration, protection, and watershed management could considerably improve the viability of the lower Fraser to support these salmon, such that many (14/19) of these populations would have a >50% likelihood of being assessed as healthy. Together, this research provides novel evidence of active and selective use of estuarine habitats by juvenile salmon, reliance on estuarine habitat for early marine growth by juvenile Chinook salmon, and a direct link between habitat health and population status for lower Fraser River salmon populations. / Graduate / 2023-04-13

Aquatic ecosystems continue to be increasingly affected by climate warming. For salmonids in the Pacific Northwest of North America, increasing temperatures pose tighter thermal constraints on their habitat use as well as aspects of their individual performance, such as disease resistance. This thesis examines the effect of temperature on the phenology of the Ceratomyxa shasta life cycle, the effect of thermal refugia on disease risk in juvenile salmonids in the Klamath River, CA, and the spatial and temporal distribution of C. shasta in the Willamette River, OR. We developed a biological model that predicts an acceleration of the C. shasta life cycle development due to climate shifts in the Klamath River, resulting in more generations per year and earlier seasonal parasite occurrence. We showed that in early summer the Beaver Creek-Klamath River confluence provides juvenile Chinook and coho salmon an area of lower parasite doses and cooler temperatures than the main stem, thus lessening disease risk. By accelerating the development of C. shasta in its hosts, increasing temperatures will result in earlier parasite transmission to juvenile salmonids and a longer season of infectivity. These fish may find disease refuge at cold tributary inflows to the main stem of the Klamath River in early summer, further adding to the benefit of these important thermal habitats. To determine if similar disease patterns occur in other rivers with the parasite, we described spatial and temporal occurrence of C. shasta in the Willamette River. By collecting weekly water sampling at four sites over 28 months we characterize seasonal and annual differences of parasite abundance, which varies with weekly temperature. We also collected samples along the length of the main stem and its tributaries and identified spatial differences in C. shasta spore densities. Identification of spatial and temporal variation of C. shasta in the Willamette River provides a foundation for understanding future patterns of disease occurrence in this river where conservation of anadromous fisheries is also of concern. This thesis identifies likely responses of C. shasta to climate warming in the Klamath River, with useful application to other rivers in the Pacific Northwest. / Graduation date: 2013

At low doses, relevant to nuclear incidents and accidental releases of radioactivity, the detriment of radiation extends beyond direct effects. This thesis investigates genomic instability, a subclass of non-targeted effects where damage and lethality is transmitted vertically and expressed in the progeny of cells many generations after initial radiation exposure. Through a series of experiments using clonogenic assay of human and fish cell culture, studies described in this thesis describe lethal mutations, hyper radiosensitivity and increased radioresistance – processes involving repair mechanisms that dictate survival in cells exposed to low doses. Further study investigates the difference in the relative biological effect of alpha particle radiation compared to what is expected at high doses. Results demonstrate increased radioresistance in a human cell line while also revealing increased lethality in a fish cell line confirming the need for consideration of dose-dependence as well as variance in behaviors of different cell lines and species. It is hoped the conclusions of this thesis will inspire the creation of protocols with greater attention to the indirect consequences of exposure to radiation at doses relevant to nuclear incidents and accidents. / Thesis / Master of Science (MSc)