Cultivation of Laminaria saccharina (Linnaeus) Lamouroux and Palmaria palmata (Linnaeus) Kuntze was trialled at three fish farm sites in north-west Scotland. Results show that seasonal yields of L. saccharina were enhanced by 50 % and P. palmata by 63 % when cultured adjacent to fish farm cages compared to environmentally similar sites away from the farms. Yields of P. palmata were further enhanced under conditions of optimal water movement. Ammonium concentrations in the seawater 0 to 50m away from the fish cages were found to be 2 - 3 μM greater than ambient. Enhanced concentrations of ammonium could be detected 200 to 300 m from the cages although the distribution is heavily influenced by local hydrography. Nitrogen content of L. saccharina and P. palmata cultured adjacent to the salmon cages in summer was higher than in seaweeds cultured at reference sites away from fish cages. Stable nitrogen isotope analysis indicates that the nitrogen in seaweeds grown next to salmon cages is derived from the fish farm and farm derived nitrogen is likely to be widely dispersed in the lochs where cages are situated. A preliminary economic analysis showed that growing seaweeds commercially, in particular P. palmata, may be at worst cost neutral, with profitability increasing as a result of enhanced production through increased nutrient availability adjacent to fish farms. A one hectare seaweed farm producing 600 tonnes wet weight over two years (300 tonnes per year) of P. palmata could potentially absorb up to 30 % of nutrients generated from a 500 tonnes salmon production unit. As farm origin nitrogen is evident in biota at distances of up to one kilometre away from the cages, cultured macroalgae would not have to be sited close to cages to result in net nitrogen removal facilitating the siting of algal farms in areas more suited for individual species requirements while still maintaining bioremediation benefits.

The growth of aquaculture industry, and in particular of salmonids, has dramatically increased in the last decades to supply the growing worldwide demand for fish products. The expansion of aquaculture needs a good management to ensure the sustainability of this activity. In particular, improving feeds for aquaculture to enhance health and performance of reared fish is imperative for this industry. Molecular biological approacches can help interpret responses to new diets and immune responses. Ubiquitin ligases (UbE3s) play a pivotal role in the ubiquitin proteasome pathway of protein degradation and are markers that could be linked to health and performance of fish. Six UbE3s were characterized, 4 are related to muscle catabolism and two involved in NF-kB activation. Atrogin-1 and MuRF genes were increased following food deprivation and a proinflammatory stimulation. MULAN and Mul1b, are mitochondrial activators of NF-kB and the expression of both genes was increased following a proinflammatory immune challenge both in vivo and in vitro. These genes have been used as markers in dietary traits to understand responses. A transcriptome study using Atlantic salmon fed a commercially available functional feed designed for optimal health status compared to a standard feed was performed. Both the liver and muscle were chosen for the transcriptomic analysis. In liver, a number of process were found down regulated, including protein turnover and innate immunity. No significant alterations were found in muscle. To determine the effect of a high plant protein diet (PP), compared to a marine profile diet (MP) in Atlantic a transcriptomic analysis was performed. Fish grew equally well on both diets and no significant histological differences were seen in intestine. The PP diet induced tissue specific changes in gene expression, with the mid intestine showing activation of the adaptive immune response and protein turnover. In liver cell proliferation and apoptosis indicate cellular reorganization and also a general suppression of processes such as immune response was observed. In contrast muscle tissue showed reduced protein metabolism and decrease in immune gene expression suggesting less energy expenditure in this tissue. To assess fish response to Piscirickttsia salmonis, the etiological agent of the salmonid rickettsial seeticemia (SRS), a transcriptomic analysis was carried out. P. salmonis infection has profound effects on transcription in the head kidney, liver and muscle of salmon. Head kidney, liver and muscle tissue showed a decrease in expression of mRNAs related to acquired immune function indicating the pathogen is down regulating this response controlled in part by the inhibition of the G-protein signalling pathway, whereas the innate immune response was increased in head kidney. Liver and muscle also responded, including specific responses in each tissue type. These results improve understanding of the mechanisms by which this bacterium survives and replicates within host cells and may assist selection of molecular biomarkers useful for the development of a diagnostic tools, vaccines and therapeutants for the aquaculture industry. Using markers selected from the previous experiments, the effect of four different diets, a marine profile diet (MP/FO), a plant protein diet (PP/FO), a vegetable oil diet (MP/VO) and PP/VO diet, in Atlantic salmon were examined. Markers for protein and lipid metabolism and immune response were studied in mid intestine, liver and muscle of fish fed for 77 days the four diets. Small but significant changes were observed, but despite of this, fish fed all the four diets showed similar growth rates. Moreover, to study the possible effect of the diets on fish health, the expression of specific SRS markers was studied in head kidney and liver of fish fed the four diets and infected with P. salmonis. The SRS challenge demonstrated that all groups of fish were able to respond efficiently to the pathogen. In conclusion, these new diets may represent good alternatives to fish meal (FM) and fish oil (FO) diets indicating that a replacement of marine diets with vegetable sources is possible in Atlantic salmon without affecting neither fish growth and performance nor the health of fish. The findings presented in this thesis suggest that E3 ligases are good molecular markers, allowing to study performance and health of fish following feeding trial and pathogen challenge. In addition, transcriptomic analysis performed to select further key molecular markers involved in a variety of processes and improved our knowledge of fish physiology and immunology, providing useful information for the development of new alternative diets for aquaculture.

The substantial increase in production by the aquaculture industry in the last few decades has been accompanied by a greater demand for prophylactic measures to control fish health, since fish diseases are a major impediment for the profitability of fish farms. To date vaccination has played a major role in the success of fish farming, offering resistance to a limited number of infectious diseases. In order to expand the availability of effective vaccines, a better knowledge of the piscine immune system is crucial. Although a tremendous effort has been put into studies of the fish immune system over recent years, our understanding is still far behind when compared to that of mammals, with many gaps in knowledge still requiring to be addressed. To gain a better understanding of fish immune responses, this thesis has focused on characterising a number of immune molecules considered crucial for disease resistance against extracellular pathogens, in a commercially important species, the rainbow trout (Oncorhynchus mykiss). Adaptive immunity is regulated by a group of specialised lymphocytes, the T Helper (T H) cells. They exert their helper functions through the release of a range of cytokines, which can be used to further categorise them into subsets. One of the latest subsets to be characterised was the T H 17 lineage, which secretes various cytokines, including interleukin (IL )-17 A and IL-22, pivotal in the eradication of extracellular pathogens. To gain a better insight into TH17-type cytokines and their responses in fish, Chapters II and III focused on the characterisation of such molecules, through expression studies, and by analysing their bioactivity as recombinant proteins for the rust time in piscine species. Homology studies confirmed that both molecules are likely to belong to the IL- 22 and IL-17 cytokine families, noting that the trout IL-17 AlP protein shared a close relationship with other piscine IL-17 AlP2 molecules. Interestingly, analysis of their biological activities in splenocyte primary cultures indicated that the two proteins had an effect on the expression of antimicrobial peptides, with IL-22 displaying a potentially more important role than IL-17 AlF. Cytokines are only able to conduct their effects through binding to specific cell surface receptors, acting as ligands. The presence of these particular receptors dictates which cells can be targeted by such cytokines and highlights the importance of the ligand-receptor interaction. To further understand the already identified cytokines, or their family members, a range of receptors was characterised in Chapter IV, with two of them (CRFB4 and IL-17RA) being potentially involved in the signalling of IL-22 and IL-17 AlF ligands. Although homology studies confirmed they belong to the IL-17 and class II cytokine receptor families, orthology of all nine receptors to mammalian homologues could not be inferred. Therefore, further work is required to achieve a better understanding of the homology of these molecules with the mammalian receptors, and their potential involvement with IL-22 and IL-17 AlF intracellular signalling. TH17 cells are characterised not only by the unique range of effector cytokines they secrete, but also through the presence of a master transcription factor, the retinoid-related orphan receptor (ROR)-yt. In Chapter V, two members of the ROR family were characterised, sharing 90% identity between each other. Trout ROR-y1 and -y2 were found to have a close relationship with known ROR-y family members, sharing a particularly high homology with the mammalian splice variant ROR-yt. This suggests that the piscine molecules are likely to be homologues of ROR-yt. However, it still remains to be elucidated whether they can drive piscine TH17 cell differentiation and indeed trigger a TH17 response in fish. T Helper (TH) cells are vital in promoting immune responses. Thus, Chapter VI aimed at developing a potentially effective antibody which could recognise the CD4 marker, typically expressed by this cell subset. The polyclonal antibody, produced using a recombinant protein approach, revealed encouraging results. It was found by Western blot analysis that this antibody could recognise proteins with a molecular weight approximating to that expected for the trout CD4 molecule and its potential splice variant. However, to confirm these observations, further work is still required t? better characterise this antibody and the cells it targets. The results of this thesis have revealed that despite the similarities between the mammalian and piscine immune systems, clear differences exist and also that fish have developed unique molecules and mechanisms not found in mammals. It is therefore of great interest to understand in more detail the role of these novel components so that future progress can be made towards gaining a better comprehension of fish immunity and help advance new vaccination strategies.