A series of investigations were carried out to assessth e intestinal microbiota of rainbow trout and the potential applications of probiotics and bovine lactoferrin (M). Farm and aquarium reared rainbow trout were examined with specific emphasis on the autochthonous microbial communities. Culture-based, culture-independent and electron microscopical investigations revealed mixed, complex microbial communities in all intestinal regions. DGGE based analysis revealed unique species present either only as allochthonous populations or autochthonous populations. 16S rRNA sequence analysis allowed species level identification of a range of isolates, many of which have not been identified from the rainbow trout digestive tract previously. Two ftirther investigations were carried out to assess the potential of using commercial probiotics and bovine Lf on growth, feed utilisation, health and intestinal colonisation of rainbow trout. Standard commercial diets were supplemented with B. subtills, B. licheniformis and Enterococcus faecium either singularly or synergistically. When comparing the findings of the joint study it can be concluded that the application of probiotics with rainbow trout, and likely other finfish species, is highly complicated. Full intestinal replacement of indigenous microbiota is not likely to be a good idea when using E. faecium; the results indicate that a synergistic relationship with the indigenous microbiota is likely to be involved in providing host benefits. Bacillus probiotics only appeared to be effective at high intestinal levels indicating that a synergistic relationship with the indigenous microbiota may not be as important. T'he joint study also indicates that it is not always possible to reproduce probiotic benefits even when using the same probionts, the same fish species and similar rearing conditions. Thus, the physiological status of the fish and the indigenous microbiota are likely to play an important role in the outcome of probiotic administration. A subsequent trial was conducted to evaluate Pediococcus acidilactic! as a probiotic for rainbow trout. The experiment was conducted to supplement the diet with either vegetative cells or lyophilised powder (as commercially provided). Despite successful intestinal colonisation, irrelevant of supplementation form few significant benefits were observed. SEM of the posterior mucosa revealed a localised colonisation pattern of P. acidilactic! between the mucosal folds similar to the observed indigenous microbiota from the farmed fish. This revelation led to a further trial to investigate the nature of probiotic colonisation through the gastro-intestinal tract using electron microscopy. The study confirmed the high colonisation of P. acidilactici on the epithelium of the anterior intestine and posterior intestine. However, it was not possible to observe such colonisation with Bacillus spp. or E. faeclum; despite culture-based results to the contrary. It is likely that the true mucosal colonisation may sometimes be confused with colonisation of the mucus layer as opposed to actual attachment to the epithelium itself. Therefore, it is crucial to utilise electron microscopy in order to confirm epithelial colonisation. The nature of both the indigenous microbiota and the application of probiotics appears to be more complicated than previously thought and continued research is clearly warranted.

Infectious Pancreatic Necrosis Virus (IPNV) belongs to the birnavirus genes, family <i>Birnaviridae, </i>and has classically been shown to be virulent to salmonid fish, including Atlantic salmon (<i>Salmo salar).</i> This study was oriented around this pathogen; it’s structural proteins in particular, namely viral proteins 2 and 3 (VP2 and VP3). Two VP2 fragments were expressed in <i>E. coli</i> and full length VP3 was expressed in <i>Pichia</i>. These proteins were tested for their abilities to confer protection against IPNV in an Atlantic salmon trial. The serum was analysed by ELISA against whole virus, and in neutralisation assays. Differences in level of response were found between the groups at the various timepoints depending on the vaccination group in all analyses. The findings from this trial suggested that VP3 had the strongest virus neutralising abilities as indicated from the 1/320 serum dilutions. VP2 has been the protein that has attracted most interest for its potential as a vaccine component. The findings of this project suggest that the importance of VP3 in protection of Atlantic salmon against IPNV has been underestimated. Another aspect of this study involved the investigation of signal sequence selection on the expression levels of the viral proteins. The outcome of this work indicated that expression of the viral proteins using the alpha mating factor signal could not be optimised. A random signal sequence library was constructed with the view to finding a novel signal(s) that was capable of yielding a higher VP3 protein expression level than that obtained by using the alpha mating factor. One sequence was discovered that was able to express VP3, which had 98% identity with the alpha mating factor signal.

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The aquaculture sector is currently thriving, and has expanded to meet the demand for fish and shellfish as an alternative protein source to meat. This is especially true for high value products such as Atlantic salmon, where in Scotland salmon farming is reported to be worth> £1 billion to the national economy. Currently around 40% of farmed fish and shellfish destined for human consumption are derived from aquaculture. Therefore, a great deal of attention is paid to problems that the industry faces, with fish diseases of paramount importance. A variety of species of bacteria, viruses and parasites are common in the aquatic environment, which can result in serious diseases amongst fish stocks. As a result, ways to improve disease resistance have been the focus of much attention, with the use of vaccines considered a desirable way forward. However, other approaches are also followed, such as the use of immunostimulants to improve fish health in a more limited, non-specific way, or the use of genetic markers to allow selective breeding of important disease resistance traits. For all of these approaches more information is needed on the pathways that give rise to disease resistance in fish in different situations, to allow their manipulation or monitoring, and the studies in this thesis are directed towards this goal. Fish has been used as a model to study the evolution of vertebrate immunolity for some deacades, especially work on humoral immune responses where knowledge on antibody production has dominated much of the literature on fish immunology. In contrast, little known about specific cell-mediated immunity in fish, even though it also likely plays an important role in the immune system and disease resistance. Therefore, this thesis has been focused on analysing such responses, taking advantage of the recently discovered cytokines of adaptive immune responses in fish, which allow transcriptomic studies in particular to look at the molecules turned on during infection and after vaccination. Thus the goal of this thesis was to take advantage of some successful vaccines that exist for rainbow trout, and examine the gene expression changes that occur in vaccinated trout post-challenge with the homologus pathogen, and to try to dissect pathways that may correlate with disease resistance in this species.

Photoperiod is an important signal involved in the timing and co-ordination of many processes such as growth and reproduction in salmonids. Both growth and reproduction appear to be controlled by endogenous rhythms, which under natural conditions, is entrained by the seasonal changes in daylength, that is accurately reflected by the diel pattern of melatonin. This thesis investigates the influence of photoperiod on growth and reproduction in rainbow trout (Oncorhynchus mykiss), and examines the effect on plasma insulin-like growth factor-I (IGF-I). These studies aim to further our scientific understanding of the endocrine mechanisms controlling growth and reproduction and transfer this knowledge to commercial trout farming practices. Studies at fry (&lt;5g) and fingerling (&gt;25g) stages investigated the effect of exposure to constant long-days (LD18:6) and constant light (LL) on growth performance. In all experiments, there was a significant growth enhancing effect following exposure to extended light regimes relative to those under ambient or constant short-day photoperiods. Growth enhancement appeared to be caused through direct photo-stimulation or a phaseshift in an underlying endogenous rhythm dependent on the timing of photoperiod application. Measurement of plasma IGF-I accurately reflected growth rate during juvenile development. Furthermore, a clear autumnal increase in IGF-I was observed that was apparently up-regulated by long-day photoperiods. Additionally, it was shown that melatonin implants significantly reduced growth rate below that of unimplanted controls irrespective of photoperiod, but did not reduce circulating IGF-I levels suggesting that elevated melatonin levels masked the perception of daylength but did not act directly on the somatotropic axis to control growth. As size or one of its correlates (growth rate, energy balance or nutritional status) is regarded as an important determinant of the ability to undergo puberty, the interaction of natural (SNP) and advancing (ADV: long-short day) photoperiods with growth, IGF-I and reproduction was investigated in virgin female broodstock. Under SNP 63% of the population attained maturity while only 29% spawned in the ADV regime. Under SNP both size and growth rate in late spring/early summer appeared to determine whether an individual may initiate maturation. Conversely, under ADV, condition factor appeared to be a better predictor. A complete seasonal relationship between plasma IGF-I, daylength and temperature was demonstrated for the first time in rainbow trout under natural conditions, and provides direct evidence for the relationship between maturation and IGF-I. IGF-I levels showed a negative correlation with testosterone as fish initiated maturation. Furthermore, IGF-I levels accurately reflected growth rate prior to the initiation of vitellogenesis, suggesting that IGF-I may provide an endocrine signal between the somatotropic and reproductive axes that nutritional status, growth rate and/or size is sufficient to initiate maturation. In addition, maturing individuals under both photoperiods typically expressed higher circulating IGF-I levels than those that remained immature and may reflect a greater opportunity for IGF-I to act on the pituitary to stimulate gonadotropin production. In this sense, the observation of elevated levels for 3 months under SNP compared to only 1 month under ADV may reflect a reduction in the window of opportunity to initiate maturation under advancing photoperiods and hence explain the reduction in fish spawning. Finally, using a commercially available ELISA kit provided evidence of a possible detection method for measurement of a leptin-like peptide as absolute levels differed significantly between mature and immature fish. This provides preliminary evidence for a possible involvement of a leptin-like peptide in rainbow trout reproduction and energy homeostasis. Given the above evidence of a growth enhancing effect of photoperiod in covered systems, the transfer of constant light (LL) regimes to uncovered commercial trout farming practices was also examined. The application of LL regimes during either fry grow-out in tanks using floating lights or ongrowing to harvest size during winter in cages using submersible lights was investigated. In all trials conducted, the exposure to LL in autumn significantly enhanced winter growth rate and feeding efficiency by up to 30% and 25% respectively relative to those maintained under ambient light. Furthermore, the effect of light intensity was clearly shown to be an important requirement in successfully applying photoperiod regimes onto the ambient photoperiod in order to enhance commercial production in &quot;uncovered systems&quot;. These studies clearly indicate the importance of photoperiod in influencing growth and reproduction in rainbow trout. Furthermore, the use of plasma IGF-I as an indicator of growth provides a practical tool for studying growth-photoperiod-reproduction interactions in this species. However, further studies are necessary to further our understanding of the endocrine pathways governing physiological mechanisms, especially growth and reproduction. Overall, this work has provided important information to improve both scientific understanding and commercial development although it is clear that substantial research is still required.