Background: The Atlantic salmon, Salmo salar L., is a prominent member of the Salmonidae family, and has been the focus of intense research because of its environmental and economic significance as an iconic sporting species and its global importance as an aquaculture species. Furthermore, salmonids constitute ideal organisms for the study of evolution by gene duplication as they are pseudotetraploid descendants of a common ancestor whose genome was duplicated some 25 to 100 million years ago. Whole-genome duplication is considered a major evolutionary force capable of creating vast amounts of new genetic material for evolution to act upon, promoting speciation by acquisition of new traits. Recently, large-scale comparison of paralogous genes in Atlantic salmon suggested that asymmetrical selection was acting on a significant proportion of them. However, to elucidate the physiological consequences of gene and genome duplications, studies integrating molecular evolution and functional biology are crucial. To this end, sequence and molecular analyses were performed on duplicated Elovl5 fatty-acyl elongases of Atlantic salmon, as they are responsible for a rate-limiting reaction in the elongation process of long-chain polyunsaturated fatty acids (LC-PUFA), critical components of all vertebrates. The aim of the research presented here was to investigate the role of gene duplication as an evolutionary process capable of creating genetic novelty, and to identify the potential ecological and physiological implications. Results: Linkage analyses indicated that both fatty-acyl elongases segregated independently and located elovl5 duplicates on different linkage groups. Genetic mapping using microsatellites identified in each elovl5 locus assigned elovl5a and elovl5b to chromosomes ssa28 and ssa13, respectively. In silico sequence analysis and selection tests indicated that both salmon Elovl5 proteins were subject to purifying selection, in agreement with previous results showing indistinguishable substrate specificities. Gene expression and promoter analysis indicated that Elovl5 duplicates differed in response to dietary lipids and tissue expression profile. Lipid biosynthesis and metabolic gene expression profiling performed in Atlantic salmon SHK-1 cells, suggested that the control of lipid homeostasis in fish is similar to that described in higher vertebrates, and revealed the particular importance of Lxr and Srebp transcription factors (TFs) in the regulation of LC-PUFA biosynthetic enzymes. Sequence comparison of upstream promoter regions of elovl5 genes showed intense differences between duplicates. Promoter functional analysis by co-transfection and transcription factor transactivation showed that both elovl5 duplicates were upregulated by Srebp overexpression. However, elovl5b exhibited a higher response and its promoter contained a duplication of a region containing response elements for Srebp and NF-Y cofactors. Furthermore, these studies indicated an Lxr/Rxr dependant response of elovl5a, which was not observed in elovl5b. Analysis of the genomic sequences of elovl5 duplicates by comparison to various sequence databases showed an asymmetrical distribution of transposable elements (TEs) in both introns and promoter regions. Further comparison to introns of the single elovl5 gene in pike indicated much higher TE distribution in salmon genes compared to the pike. Conclusions: Although not conclusive, the most parsimonious origin for the salmon elovl5 duplicates is that they are derived from a WGD event. This conclusion is also supported by the close similarity of two elovl5 paralogs in the recently available rainbow trout genome. Regardless of their origin, Atlantic salmon elovl5 genes have been efficiently retained in the genome under strong functional constraints indicating a physiological requirement for both enzymes to be functionally active. In contrast, upstream promoter regions have strongly diverged from one another, indicating a relaxation of purifying selection following the duplication event. This divergence of cis-regulatory regions has resulted in regulatory diversification of the elovl5 duplicates and regulatory neofunctionalisation of elovl5a, which displayed a novel Lxr/Rxr-dependant response not described in sister or other vertebrate lineages. Promoter analysis indicated that the observed elovl5 differential response to dietary variation could be partly attributed to varying transcriptional regulation driven by lipid-modulated TFs. The distribution of TEs in elvol5 genes of Atlantic salmon shows a clear increase in TE mobilisation after the divergence of esocids and salmonids. This must have occurred after the elongase duplication and thus the salmonid WGD event and contributes to the observed regulatory divergence of elovl5 paralogs.

Current feed formulations within the aquaculture industry have tended to rely on high dietary lipid thus offsetting relatively expensive protein as a source of energy. In this way, protein can be ‘spared’ for synthesis of new tissue and the high lipid content can also fulfil both fish and consumer essential fatty acid (EFA) requirements. However, the main disadvantage of feeding high lipid levels to farmed fish is a surplus of fat deposition in the flesh and other important tissues, which can detrimentally impact on quality characteristics central to the human consumer. However, based on previous work in other animal models, it is entirely feasible that supplementation of the diet with bioactive fatty acids such as conjugated linoleic acid (CLA) and tetradecylthioacetic acid (TTA) may mitigate the deleterious effects of feeding farmed fish high fat diets by reducing fat deposition in particular. The general objective of this research work was to test the hypothesis that CLA and/or TTA could augment growth, reduce fat deposition and enhance fatty acid composition via incorporation of these bioactive fatty acids, and increase n-3 highly unsaturated fatty acid (HUFA) levels in the flesh of commercially important fish species such as Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua L.) and rainbow trout (Oncorhynchus mykiss). This project also considered the influence of CLA and TTA on enzymes and transcription factors thought to be pivotal in lipid metabolism and fatty acid oxidation in particular. A subsidiary aim of this research work was to investigate the immunological impact of dietary CLA and TTA administration in these fish. The results of this project have revealed that the hypothesis was only partly proved. There was no effect in growth or biometry after either CLA or TTA supplementation in any of the fish species investigated. Additionally, there were few physiologically significant effects on fat levels on fish as a result of TTA or CLA administration. However, there were a number of effects on fatty acid metabolism including inhibition of steroyl coenzyme desaturase (SCD) in cod and trout in particular and also enhancement of hepatic n-3 HUFA levels in trout. Importantly, it was determined that both TTA and CLA could be incorporated into the flesh thus providing a vehicle through which these bioactive fatty acids can be delivered to the consumer. There were also a number of beneficial effects on activity and gene expression of a number of enzymes and transcription factors thought to be fundamental to the modulation of fatty acid oxidation in particular. However, the effects on gene transcription and biochemistry had little impact at the whole body level. This research work also showed that there were no detrimental effects on immune status after supplementation with dietary CLA or TTA. Conclusively, this thesis has contributed to the overall understanding of the influence of dietary CLA and TTA in farmed fish.

Indiana University-Purdue University Indianapolis (IUPUI) / An investigation of the lipid metabolism pathway in the yeast Yarrowia lipolytica was conducted. Yarrowia is an oleaginous ascomycete that is capable of growing on many different substrates, which derives its name from its high efficiency of growth on lipids. Once the exogenous lipids are converted into free fatty acids and internalized by the yeast, the primary mode of degradation is through β-oxidation mediated by the peroxisomal oxidases, or POX genes. These enzymes catalyze the formation of a trans double bond, producing the trans-2-enoyl product. Our study looked at the comparison of the Y. lipolytica prototrophic strain against a knockout of the Pox2 gene on the uptake, incorporation, and degradation of relevant fatty acids. To construct this gene knockout, a novel gene deletion method using a combination of Cre recombinase and the AHAS* gene was synthesized, developed, and tested successfully. This knockout system allows for serial deletion of genes with the use of only one resistance marker, with excision of the marker after selection.