Aquaculture is known as a major food-producing industry and Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) are the major cultured species in Scotland. However, disease outbreaks in aquaculture have been reported and are commonly associated with intensive fish farming, which results in a tremendous cost in the industry. Hence, understanding what immune-related genes and cells are present, their responses, mechanisms and functions in these farmed animals is a first requirement for potent vaccine design, selection of disease-resistant breeds and disease outbreak prevention. The innate immune system is the first line of defence against microbes which use germline-encoded pattern recognition receptors (PRRs) to recognise specific, conserved and constitutive products of invading pathogens, called pathogen-associated molecular patterns (PAMPs) that are important for survival of the microorganism and are thus hard for the microorganism to change. This thesis focuses on the identification and characterisation of a family of PRR called toll-like receptors (TLRs) and a TLR accessory protein UNC93B1 using different approaches. In Chapters 2, 3 and 5, eleven TLR genes and UNC93B1 were identified from Atlantic salmon whole-genome shotgun (WGS) contigs. These genes were cloned and sequenced and their putative domain structure, gene synteny and homology to other genes were determined by bioinformatics analysis. In addition, the constitutive expression profile of these genes was examined in different tissues from healthy salmon using real-time PCR. The potential modulation of these genes was examined in different in vitro and in vivo models which provide information to help understand the role(s) of these genes during inflammation or in the immune responses against pathogens. Several of these TLRs are so-called non-mammalian TLRs (TLR19, TLR20a and TLR20d) and are therefore particularly interesting to study. The sub-cellular localization was also investigated in TLR-GFP expression plasmid transfected Salmon Head Kidney-1 (SHK-1) cells. Lastly, attempts were made to develop a Human Embryonic Kidney (HEK) 293T cell line based platform to study TLR signalling and ligand specificity (Chapter 4).

The present study investigated spatial and temporal relationships between fluctuations in the abundance of Atlantic salmon (Salmo salar L.) and plankton records from the Continuous Plankton Recorder (CPR) in the North Atlantic Ocean. Analyses that examined co-variation in time series of salmon pre-fishery abundance (PFA) from fishing nations on both sides of the North Atlantic revealed a high degree of common variation between neighbouring nations for abundance of one sea winter salmon (1SW) and a common decline in the long-term abundance trends for multi-sea winter salmon (MSW). An appraisal of the data attributes for a selection of plankton taxa sampled by the CPR, corresponding to seasonal abundance in specific regions, indicated that these data capture useful spatial and temporal information on the abundance of the sampled taxa. Spatial interpolations were carried out using Data-Interpolating Variational Analysis (DIVA) for planktonic taxa that have been proven to relate to Atlantic salmon in previous studies, namely Calanus finmarchicus, Euphausiacea and Hyperiidae. Geographical boundaries were established for distinct populations of Calanus finmarchicus and for distinct aggregations of species in the cases of the Euphausiacea and the Hyperiidae. This knowledge was enhanced with information on the likelihood of persistence of these populations obtained by analysing the coherency of their seasonal cycles of abundance. This study detected strong relationships, in the form of long-term correlations, between the three selected plankton taxa (Calanus finmarchicus, Euphausiacea, Hyperiidae) and the diatom component of phytoplankton. These results indicated that the population dynamics of plankton species assemblages in these regions are influenced by common biotic and abiotic factors. Significant relationships were found between pre-fishery abundance PFA estimates for post-smolt salmon and Hyperiid amphipods. The relationships detected were particularly strong for 1SWsalmon of British and Icelandic origin at a lag of 1 year, i.e. corresponding to effects on the early phase of migration at sea. Relationships detected corresponding to the Euphausiacea and Calanus finmarchicus were more prevalent at lag-0 after the first winter at sea i.e. they relate to effects on the adult salmon. The results corresponding to MSW salmon also provided some indication of carry-over effects from the early phase of migration on adult survival. This work provided support for the hypothesis that early marine mortality is critical to the overall dynamics of salmon populations. Such information on ecosystem interactions is needed to improve the current knowledge on factors relating to salmon survival at sea. This information can be used to fine-tune important parameters of predictive models for stock management or conservation of salmon, especially in the face of the pressures of climate change.

The aim was to develop new methods for measuring texture of Atlantic salmon (Salmo salar L.) fillets and investigate the genetic basis of flesh quality traits. Firstly, a new tensile strength method was developed to quantify the force required to tear a standardized block of salmon muscle with the aim of identifying those samples more prone to factory downgrading as a result of gaping. The repeatability, sensitivity and predictability of the new technique was evaluated against other common instrumental texture measurement methods. Data from the new method were shown to have the strongest correlations with gaping severity r=-0.514, P<0.001) and the highest level of repeatability of data when analysing cold-smoked samples. The Warner Bratzler shear method gave the most repeatable data from fresh samples and had the highest correlations between fresh and smoked product from the same fish (r=0.811, P<0.001). It is therefore recommended that the new method be adopted for measuring gaping potential and the Warner Bratzler method become the standard for firmness assessment. Genes associated with post mortem softening in mammals were characterised in Atlantic salmon. A previously unknown ancient paralogue of calpastatin (here named CAST2) was identified. Evidence was provided for the existence of highly homologous recent paralogues of CAST2 and CTSD1. Evidence for the ancestral history of these paralogues was provided by phylogenetic analysis. Recent gene duplicates of 6 further genes were identified. In all cases, homology between recent paralogues was greater than 94%. Analysis of synonymous vs non-synonymous nucleotide substitution between the observed paralogue pairs shows a significant purifying selection in most cases. The CTSD1 gene shows significant purifying selection in a pairwise analysis between 12 teleost species (all cases P<0.0001) but a similar analysis of CTSD2 revealed no significant occurrence of purifying selection. The present study provides further support for the idea of asymmetrical selective pressure on paralogues. Genetic markers were developed that can distinguish individuals with above average fillet yield and texture. A database of firmness, tensile strength and fillet yield was made from 254 individuals from 5 batches of farmed salmon and these fish were genotyped at 7 novel SNP loci. Individuals with the combined favourable genotype at CAPN1a and MYOD1b were associated with an average increase in fillet yield of 2.7% above batch average. A combined genotype of CAPN1a, MYOD1b and MYF5 was significantly associated with an average increase in tensile strength of 9.8% above batch average (P=0.015). In both cases individuals with the combined favourable genotype occurred with a frequency of c. 6% across all batches. The favourable genotypes had no unfavourable effects on other traits. Highly polymorphic microsatellite loci were used to perform tests of assignment, which revealed an overall correct assignment rate of 92.7% to batch of origin and a minimum reference sample number of 25 was empirically determined. A phylogenetic analysis supported the results of the assignment tests. Given that 7 microsatellites is a relatively small number for a study of this nature, these results suggest that reliable assignment of unknown fish to the true batch of origin is potentially rapid and cost effective. Overall, the thesis presents molecular markers for broodstock selection, new genes of relevance to flesh quality, a new method of texture analysis and a proposal for an escapee traceability project.

Atlantic salmon, SaI1M salar. L., have been shown to be more susceptible to infections by the caligid copepod Lepeophtheirus salmonis (Kreyer, 1837) than other salmonid species. Atlantic salmon exhibit a reduced cellular immune response to the attached parasite which has led to the hypothesis of the presence of sea louse associated compounds that depressed the fish's normally efficient inflammatory mechanisms. The aim of the current study was to test this hypothesis. A biochemically defmed in vitro culture system was developed that would allow collection of the secreted/excreted products of the copepodid larvae of L. salmonis, and avoid their contamination by metabolites of the host from their collection, in vivo. Available tissue culture methods proved inadequate in supporting copepodid culture because of the louse requirement for a seawater maintenance medium that was osmotically unsuitable for cultured cells. Tissue engineering technologies developed in the construction of human living skin equivalents were successful in the development of an Atlantic Salmon Skin Equivalent (ASSE). ASSE is a novel organotypic tissue culture substrate that was constructed from Atlantic salmon fibroblasts (AS-6) and primary cultures of Atlantic salmon epithelial cells. Cells were supported in a matrix of collagen fibres, acid extracted from the tails of rats, and combined using a layering technique to create a substitute salmon skin. ASSE has a fibroblastic dermal equivalent overlaid by an epidermis-like layer and a layer of collagen, and was maintainable in a seawater media. During its development, the cells within ASSE showed signs of differentiation that included stratification, increased fibronectin production by cultured fibroblasts, and the formation of a basement membrane-like layer at the junction of the dermis and epidermis. In this environment, ASSE allowed the survival of copepodid larvae for an average of 12 days, a period approximately 5 days longer than that of their free-swimming counterparts. Furthermore, cultured copepodids were observed feeding, exhibiting the normal range of settlement behaviours, and also showed increases in their length. However, metamorphosis to the chalimus I stage did not occur and was not stimulated by the supplementation of ASSE with salmon mucus, salmon peptone or DL methionine. Nevertheless, copepodid maintenance on ASSE allowed the collection of the compounds they released into the culture media. These were assayed for their effects on the immunological functioning of salmon macrophages. These assays demonstrated that the chemotaxic ability of macrophages was significantly reduced following treatment with louse culture supernatants . (LCS), as was their phagocytic ability. In both assays, the activity of each function was approximately 50% lower than that recorded in untreated cells. Intracellular respiratory burst and the phagocytic index of active phagocytes, however, was not affected. The biological activity of LCS was inhibited following heating, dilution, and treatment with proteinase K, indicating that the active immunosuppressive compounds were either themselves proteins, or required protein to be effective. The active substance was designated Louse Immunomodulatory Factor (LIF). The enzyme profiles of culture supernatants were investigated using API ZYM test strips. The profiles of LCS supernatants were significantly different to those of control supernatants from the early stages of louse incubation with ASSE. LCS supernatants showed elevated levels of leucine aminopeptidase, C4 and C8 esterases, alkaline phosphatase, P-glucuronidase, and Nacetyl- j-glucosaminidase. The involvement of these enzymes in the digestive processes of arthropods is well documented. However, the presence of the chitin hydrolysing enzyme Nacetyl- Beglucosaminidase, may also be suggestive of the preparation of copepodids for moulting. This, and the involvement of these enzymes in digestion in copepodids, and as possible immunomodulatory compounds is discussed. Gel filtration chromatography identified 14 proteins in the LCS that were not present in the control supernatants. These proteins were in the molecular weight range <1 kOa to 2665 kOa. No biological activity was attributed to these proteins when isolated by chromatography and assayed for their effects on macrophage chemotaxis. This lack of activity may be associated with their dilution during the chromatography process in which the supernatants were diluted approximately 5000 times. Experimental evidence showed that the activity of LIF was absent when supernatants were diluted to 1:1000, and so methods of concentration may be required in order to establish the immunological activity of these proteins. This study has developed an Atlantic salmon skin substitute that could have a great number of applications in the study of salmonid metabolism, cellular communication, immunology, and drug and chemical testing. Here, it supported the extended maintenance of sea lice larvae in vitro and allowed the collection of the products of their culture. The findings have shown that the copepodid stage of L. salmonis produces substances, LIF, that depress the chemotaxic and phagocytic activity of salmon macrophages, in vitro. These active substances may also be responsible for the depression of the inflammatory responses of sea lice infected Atlantic salmon, in vivo.

The object of this thesis was to investigate the role of two different harvest protocols on the post mortem physiology of Chinook salmon, and associated deteriorative processes that occur during frozen storage of the white muscle tissue. The two different harvest methods employed, termed 'rested' and 'exercised', were selected because of the contrasting levels of activity of the animal prior to, and upon, slaughter. While the latter represents conventional harvest techniques Rested and exercised harvesting protocols produced tissue in significantly different physiological states. Immediately post harvest, rested tissue maintained high metabolic energy stores of ATP and glycogen within the tissue, with low concentrations of tissue and plasma lactate. Exercised tissue exhibited near depleted concentrations of ATP and glycogen and a marked metabolic acidosis and lactate accumulation. When frozen immediately post harvest, rested white muscle tissue stored at -19℃ showed no significant changes in these metabolite concentrations over a six month period of profiling. However, during storage of rested tissue at -9℃, hydrolysis of ATP and glycogen with no coincident increase in lactate was observed. No significant changes in metabolite levels were observed within exercised tissue stored at -19 and -9℃, owing to the lack of metabolic energy stores. Transfer of tissue from frozen (-80 and -19℃) to chilled (-1 and +4℃) temperatures witnessed a rapid depletion of tissue ATP and glycogen stores, with rapid increases in tissue lactate concentrations. This metabolic activity was more significant in rested tissue owing to the larger concentrations of metabolic energy stores. This metabolic activity was identified to occur between the temperatures of -3 and -1.5℃ and occurred abruptly (i.e. ATP concentrations depleting in less than one hour) in time. During frozen storage (-19℃ and -9℃), harvest treatment had no significant effect on lipid oxidation processes. However, rested tissue showed a significant ability to retard lipid oxidation processes once removed from frozen storage and placed at chilled temperatures. Throughout six months storage at -19℃ storage, harvest treatment had a significant effect on the rate of protein denaturation as rested tissue consistently held higher concentrations of soluble protein over the storage period. No significant effect was observed between treatments in the rate of protein denaturation during one month frozen (-19℃) then chilled (+4℃) storage. In a supplementary frozen (-80℃) then chilled (-1℃) storage experiment, post mortem storage of rested, whole fish, at chilled (+5℃) temperatures prior to white muscle excision and freezing, was compared to rested and exercised tissue in which the white muscle had been excised and then frozen immediately post harvest. In this experiment rested tissue exposed to a 6 or 24 hour post mortem chilled storage period demonstrated significant retardation of lipid oxidation processes when compared to rested white muscle tissue that was excised and frozen immediately post harvest. Further comparison of the six and 24 hour post mortem stored tissue showed a significant increase in lipid oxidation products after 21 and 24 days chilled storage, respectively. Comparison of results from the six and 24 hour post mortem storage experiment were bordering on significance (p=0.083), warranting further investigation on the effect of post mortem storage of rested tissue on lipid oxidation processes.

<i>Caligus elongatus </i>and <i>Lepeophtheirus salmonis </i>are ectoparasites of Atlantic salmon and sea trout (<i>S. trutta).</i> Solid phase extraction (SPE) and stir bar sorptive extraction (SBSE) were used to investigate and compare the odour profiles of Atlantic salmon, sea trout, haddock and turbot. The behavioural responses of <i>L. salmonis </i>copepodids towards the different extracts of the four species were tested on Y-tube bioassays. Copepodids showed significant increases in swimming activity in the presence of SPE and SBSE extracts of salmon and sea trout. The copepodids showed a significant preference for SPE extracts of salmon and sea trout only. Haddock SPE extracts elicited a significant increase in activity but no significant odour preference. <i>L. salmonis</i> copepodid behaviour when presented with sea trout and haddock odours was tested in a Y-tube bioassay. Significant increases in activity in the presence of both species odours was found. Copepodids showed a significant preference for sea trout conditioned water over blank artificial seawater and for artificial seawater over haddock odour. <i>C. elongates</i> copepodids showed no significant chemotactic responses in Y-tube experiments. Analysis of video tracked three-way olfactometer experiments found significant increases in the swimming activity and larger turning angles by <i>C. elongates </i>in the presence of salmon odour. When compared to <i>L. salmonis, </i>significant differences in swimming behaviour were found. A weak but significant correlation was found between the ecology of the hosts and the presence or absence of Caligid copepods. This is discussed in the context of the ecological traits and the influence these have on potential co-occurrence. In addition the reproductive strategies of <i>C. elongatus</i> and <i>L. salmonis</i> were investigated and the differences found are discussed in the context of co-existence.

This research investigated the ecological significance of GW-SW interactions in the hyporheic zone of two Scottish mountain rivers (the Girnock Burn and the River Feshie).  The research comprised three components: (i) an assessment of the influence of GW-SW interactions on the spatio-temporal variability of hyporheic hydrochemistry and macroinvertebrate community structure, (ii) an evaluation of the influence of GW-SW dynamics on small-scale spatio-temporal variability in hyporheic thermal regimes, and (iii) an investigation of the spatial relationship between Atlantic salmon <i>Salmo salar </i>(L.) spawning distributions and patterns of GW-SW exchange in a braided river reach. In the Girnock, marked inter- and intra-reach differences in hyporheic water quality were found and linked to spatio-temporal variability in GW-SW exchange.  Up to 25% of the spatial variability in invertebrate community structure could be accounted for by differences in hyporheic water quality.  Community composition varied markedly between winter, spring and summer seasons, with the relative importance of individual water quality variables differing between seasons. There was much heterogeneity in streambed temperature regimes across the study reaches, with data suggesting that long-residence groundwater contributed significantly to hyporheic exchange.  However, at small (cm to m) scales thermal variability was most likely explained by the interaction of reach-scale GW-SW exchanges with smaller-scale, current-bed form induced hyporheic exchange. In a braided section of the River Feshie, spatially complex and temporally dynamic GW-SW exchange patterns occurred.  Salmon spawning was concentrated in locations characterised by upwelling groundwater at depth, with up to 66% of spawning sites recorded in groundwater dominated channels.

The interest in sports fishing and the number of enterprises involved is a growing phenomenon in Sweden. According to statistics from Statistics Sweden, about 1.6 million of all Swedish residents has an interest in fishing and about 10 percent of all international trips are related to fishing tourism. This indicates that there is a potential to develop fishing tourism in Swedish rivers. The aim of this study was to explore the fishing tourism around the salmon fishery and to identify any action that has been taken to improve conditions for the salmon in the river Göta älv, in the area of the Lilla Edet and Trollhättan municipalities. The information in this study has been collected from literature and interviews with SFK Laxen and Trollhättan Municipality. Göta älv is, among other rivers, regulated for hydropower production with four hydropower dams blocking the migration routes for the salmon. The main measurements that have been taken to facilitate salmon migration and improve conditions for salmon fishing are fish ladders at the power stations, and the addition of gravel to create spawning grounds. However, many projects that would improve the potential for fishing tourism are still waiting to be approved or has been denied. The result of this study has clarified the current situation for salmon and salmon fishing in Göta älv, and make some suggestions for further development of fishing tourism in the river.

Thesis advisor: Noah P. Snyder / Substrate size and mobility are important to Atlantic salmon (Salmo salar) spawning and rearing success. Channel geometry is a control on bedload mobility in streams. It is believed that channel morphology in many Maine rivers has been altered by land use practices, creating wider and shallower channels, and lowering stream competence. If correct, these changes may be partially responsible for the limited number of returning salmon currently observed in Maine coastal rivers. To evaluate the magnitude of these changes, I performed a statistical comparison of channel morphology between two Atlantic coastal streams: the Narraguagus River in Downeast Maine and the Jacquet River in northern New Brunswick, Canada. Compared to the Narraguagus River, the Jacquet River has relatively healthy returns of adult salmon. Both watersheds have similar drainage areas (Narraguagus 588 km²; Jacquet 510 km²;) and mean annual precipitation (1244 mm; 1200 mm), but differing average channel gradients (0.16%; 0.51%) and longitudinal profiles. During the summer of 2007, I surveyed a 13.6-km section of the Narraguagus with a drainage area range of 129-247 km², and a 10.4-km section of the Jacquet with a drainage area range of 94-265 km². I made measurements of active and bankfull width and depth, and channel gradient at 100-m intervals, and performed grain-size counts at 200-m intervals. I also measured gradient and width in a GIS-based analysis. Results of my analysis show that channel gradient is likely the most influential factor on Atlantic salmon habitat as it relates to sediment size. The two rivers exhibit no significant difference in width-to-depth ratio, when low-gradient outliers in the Narraguagus River are removed. Predicted median riverbed grain sizes were calculated using two methods: (1) from the empirical relationship between basal shear stress and measured grain size; and (2) using the Shields parameter and remote sensing data only. Measured and predicted grain sizes reveal finer river-bed sediments on the Narraguagus River, however, Shields parameter calculations show that sediment should be mobile in both streams. I compare these predictions to field-based habitat mapping on the Narraguagus River. Based on predicted grain sizes, I expect nearly continuous Atlantic salmon spawning (28-95%) and rearing (95-100%) habitat on the Jacquet, and much less spawning (47-62%) and rearing (57-68%) habitat on the Narraguagus. This is likely because the Narraguagus River is segmented into reaches of steeper gradient (S < 0.002) with potentially good habitat, and flatter reaches (S < 0.0005) of poor habitat. The long flat reaches (several km) likely act as sediment sinks, preventing the continuity of downstream sediment transport and causing sediment to be sourced from localized glacial deposits. / Thesis (MS) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Geology and Geophysics.

Thesis advisor: Noah P. Snyder / Dam-influenced streambank morphology has not been studied extensively in rivers in deglaciated landscapes with high densities of colonial-era milldams. Fluvial restoration in the eastern U.S. often focuses on understanding pre-Colonial floodplain processes. Recent work by Walter and Merritts (2008a) in the Piedmont of the U.S. mid-Atlantic region suggests milldams significantly impact sedimentation by creating surfaces composed of post-dam legacy sediment that are often abandoned by the river and function as fill terraces. I analyze channel morphology and sedimentation patterns upstream of two breached dams on the Sheepscot River in mid-coastal Maine using lidar digital elevation models, historical aerial photographs, radiocarbon dating, and hydraulic modeling. In the past several decades, observable channel morphologic changes occurred at the two study sites: Maxcy's Mills dam (built in 1809, it was 2-m high and breached in the late 1950s), and at Head Tide dam (built in the 1760s, it is 4-m high and was partially breached in 1952). The Sheepscot River has a native population of Atlantic salmon, which is a federally listed endangered species. Understanding the existence and transport of legacy sediment has become an important component of habitat restoration efforts in the region. The goal of this investigation is to determine the extent and morphologic function of legacy sediment in order to better understand how historical dam sites affect channel morphology and sediment transport in a post-glacial, low-gradient river system. Field and remote sensing analyses indicate that surfaces (up to 2.65 m high) composed of mud and sand function as floodplains 1 km upstream from Head Tide dam and surfaces 90 cm high continue 2.5 km upstream from Maxcy's Mills. Analysis of seven radiocarbon dates from pieces of tree bark sampled from the stratigraphy (58-187 cm below the surface) of the two study sites suggest up to 1.8 m of sediment upstream of the two study sites was deposited within the past 300 years and is therefore a legacy of the dams. Quantification of the total volume of stored legacy sediment is on the same scale as volumes observed in the mid-Atlantic Piedmont region, leading to the conclusion that post-glacial rivers in northern New England store milldam sediment in similar fashion to streams analyzed in the Walter and Merritts (2008a) study. / Thesis (MS) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Earth and Environmental Sciences.