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Numerous populations of wild Atlantic salmon have declined in recent years. The Atlantic salmon in Girnock Burn, an upland spate tributary of the River Dee, Scotland have been monitored intensely by government scientists since 1966. The burn is equipped with upstream and downstream traps, which have enabled monitoring of juveniles leaving the burn and adults returning to it since 1966. Recently, due to a decline in numbers of female returns, a supportive breeding program was instigated. Using microsatellite-based DNA profiling, this study exploited existing and novel tissue samples to investigate aspects of Atlantic salmon biology and conservation. A panel of up to 12, mainly highly polymorphic, microsatellite loci were employed to derive allele frequency data and to resolve parentage in egg, parr, smolt and anadromous adult samples taken between 1991 and 2004. Genotyping error was investigated and rectified where possible. Overall, the detected error was low (c.0.5%), providing confidence in subsequent population and parentage analyses. The error rate involved in estimating the age of salmon in Girnock Burn from scale readings was also estimated (c.2-8%). A study of the dynamics of natural spawning, based on the parentage of parr, confirmed that multiple matings by anadromous returns of both sexes were prevalent. Not all anadromous returns were apparently successful spawners; data from parr and existing redd samples failed to detect a contribution from 35% of males and 29% of females. An important aspect of the work was to determine the success of the supportive breeding program. Results showed that, in comparison to natural spawning, the program gave a more complete and even representation of adult spawners in offspring. In addition, there was no detectable difference in the output (number of smolts) of the two schemes when the number of eggs used in each was taken into account. The distribution of juvenile kin (parr aged 1+) within the burn was determined, which revealed clustering of full and half sib groups. This was found to impact on standard population genetic analyses. Adjacent samples (n = 50), each sampled over a c.1.5 km stretch of river were shown to exhibit significant allelic differentiation, while samples from individuals selected at random over a 7.5km stretch did not. Parentage analysis of adult returns showed that the number of returns likely to be philopatric was higher than would be predicted solely from physical tagging data. This was attributed to ‘leakage’ of the downstream parr/smolt trap. An initial investigation into the role of mature parr in adaptation of populations to the environment was made, although sire type (i.e. anadromous male or mature parr) was not found to affect survival in the freshwater environment in this case. More research into this aspect is warranted, particularly with the possible impact of predicted climate change on male parr maturity. A comparison of genetic diversity through time (measured by allelic richness) revealed no detectable change between 1991 and 2004. Estimates of the effective population size using different genetic (temporal) methods were associated with a large degree of uncertainty, and were surprisingly high (ranging from 595 to 1992) c.f. demographic based estimates (ranging from 95 to 144), which was likely to be due in part to violation of assumptions made in the calculations. These findings have highlighted a range of avenues for future lines of research, should aid in the management of Atlantic salmon within Girnock Burn and assist in the design of sampling regimes.

Fish meals (FM) and fish oils (FO) are used extensively as the main protein and lipid sources respectively in industrially compounded salmon feeds, mainly due to their excellent nutritional properties. Nevertheless, several reasons dictate the utilisation of sustainable alternative protein and lipid sources and the subsequent reduction in the reliance on fishery-products in aquafeeds. Soy protein concentrate (SPC) is a very promising alternative to FM Hence, the main objective of the present thesis was to investigate the effects of the increased substitution of FM withSPC, lysine and methionine on the growth performance and immune responses of early and late stage Atlantic salmon parr prior to and after vaccination with commercial vaccines (Experiments II and V presented in Chapters 4 and 6). Furthermore the effects of increasing dietary levels of SPC with consistent and/or increasing dietary supplementation of phosphorus on the Atlantic salmon parr’ body proximate and mineral composition or the skeletal mineral composition respectively, prior and post-vaccination with commercial vaccines; under continuous light or under 12 hours light: 12 hours dark photoperiod (Experiments I and V presented in Chapters 3 and 5 respectively). Lastly the impact of FM-, FO- and fishery-free diets on the growth, carcass proximate composition and immune status of Atlantic salmon post-smolts was investigated (Experiment III presented in Chapter 7). In the latter experiment six diets were tested including: two commercially applied marine based diets, one with partial inclusion of vegetable proteins (VPs) and oils (VOs) according to the EU standards (2011-12) (MBE) and one with partial inclusion of VPs, VOs and land animal-by product (ABP) proteins according to the non-EU standards (MBABP); a fully vegetable protein (VP) diet; a fully algal and vegetable oil (VO) diet; a marine-free VP and VO and algal oil diet (VP/VO) diet; and a marine-free diet with a mix of VPs and land ABP proteins and lipid from VOs and algal oils (MFABP). The results of the Experiments I and II (Chapters 3 and 4) show that late Atlantic salmon parr can grow efficiently on SPC80 diets, however, they require longer periods to adapt to these diets compared to fish fed diets containing lower levels of SPC as a protein source. Decreased mineralisation of body cross-section was observed for salmon fed increasing dietary SPC. Vaccination improved mineralisation for the high dietary SPC salmon groups. However, continuous light exposure promoting fast growth appeared to be detrimental for Atlantic salmon body cross ash, Ca, Mg, Mn, P and Zn. Moreover, it was demonstrated that substitution of up to 50% of high quality FM protein with SPC and constantly added P has the minimum possible impact on late salmon parr growth, whereas it stimulates several immune parameters prior to immunisation. Immunostimulatory effects were also shown for the diets with higher dietary SPC levels. It is not clear if these results were an effect of increased FM replacement with SPC or not properly balanced levels of dietary P. The Experiments III and IV (Chapters 5 and 6) illustrated that early stage Atlantic salmon parr can accept diets with up to 58% protein from SPC without serious effects on body growth. However, higher levels can severely affect salmon growth performance. Moreover, it was shown that long-term feeding of salmon with increasing dietary SPC combined with increasing phosphate supplementation, alone or in combination with vaccination can actually be beneficial for Atlantic salmon parr bone mineralisation. However, mineralisation in vaccinated fish was higher than in PBS-injected fish. This could be linked to the slower growth of vaccinated salmon allowing their developing bones to mineralise properly. Changes in the modulation of the different components of the complement activity was revealed in Atlantic salmon fed increasing dietary levels of SPC. The modulation of complement activity was demonstrated at both studies utilising increasing dietary SPC concentrations, indicating that complement componenets are among the most prominent immunological markers upon dietary FM replacement with SPC. However, overall no differences in total complement activity and therefore the immune capacity and resistance against Aeromonas salmonicida were observed among the salmon groups receiving increasing levels of SPC. Lastly in Experiment V (Chapter 7) higher growth performance indices (weight gain, SGR and TGC) were evident in the MBE salmon compared to the MBABP group, salmon fed diets with complete eleimintion of FM or FO (VP and VO respectively) and fish fed two diets with total substitution of both marine derived feed ingredients, three months after the start of the feeding trial. Higher feed intake was demonstrated for both MBE and VP salmon compared to the other groups for the duration of the first period. The above results could have been influenced by discrepancies in the size of the fish at the start ofthe trial, revealing flaws in the experimental design. Both MBE and VP salmon groups also presented the highest feed conversion ratios, revealing the lowest efficiency in dietary nutrient utilisation in comparison to the rest of salmon which exhibited no differences in feed efficiency, revealing an overall better performance of the MBABP and diets with low levels of marine feedstuffs. Improved FI compared to the values of the first period and higher SGR and TGC values were demonstrated for salmon from the latter treatments compared to MBE and VP salmon, during the second part of the study, revealing compensatory growth for these groups. VP salmon demonstrated the highest and VO salmon the lowest condition factor values. The former finding might possibly be related with higher fat accumulation in the viscero-hepatic tissues. No differences were observed in carcass moisture, protein, fat and ash concentrations among the dietary groups of fish. Furthermore, no differences were demonstrated in terms of total and differential leucocyte counts, plasma haemolytic activity, plasma protein and total IgM levels, stimulated and non-stimulated HKM burst activity among the different dietary groups. However, lower haematocrit values were observed in the MB and VO-fed groups compared to the MFABP and VP/VO groups. Furthermore, decreased lysozyme activity was observed for all diets in contrast to the control groups, whereas FM-free diets promoted plasma anti-protease activity. The former result could have been an effect of either immune or stress induction, whereas the second is regarded as an immunostimulatory effect. The results suggest that marine-oil, marine-protein and marine-free diets could be satisfactorily used for Atlantic salmon post-smolts without severe reductions in their innate immune responses, although longer adaptation periods might be required for the fish to fully accept these diets.

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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.

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.

<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.