Author: Melissa Kate Gregory
Gregory, Melissa Kate, 2010 Investigation of the long chain polyunsaturated fatty acid synthesis pathway in Southern Bluefin Tuna (Thunnus maccoyii) and Yellowtail Kingfish (Seriola lalandi), Flinders University, School of Biological Sciences
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The essential polyunsaturated fatty acids (PUFA) for humans, 18:3n-3 (á-linolenic acid, ALA) and 18:2n-6 (linoleic acid, LA), must be obtained through the diet because they cannot be synthesized. Humans consume a diet rich in n-6 fatty acids and are not able to convert the essential dietary n-3 PUFA ALA into n-3 long chain polyunsaturated fatty acids (LCPUFA) like 20:5n-3 (eicosapentaenoic acid, EPA), 22:5n-3 (docosapentaenoic acid, DPA) and 22:6n-3 (docosahexaenoic acid, DHA). In contrast to humans, fish are considered to have a functional LCPUFA synthesis pathway which can convert ALA into the LCPUFA derivatives EPA, DPA and DHA. The vertebrate LCPUFA synthesis pathway requires three elongation and three desaturation steps to convert ALA to DHA. The fatty acyl Ä6desaturase and fatty acyl elongase, Elovl5, are both considered to be used twice. This thesis aimed to examine the LCPUFA synthesis pathway, in particular Elovl5 and Ä6desaturase, in freshwater, anadromous and marine fish species. Three fish models were used to examine the accumulation of individual PUFA and their subsequent LCPUFA products. Yellowtail kingfish (YTK; Seriola lalandi) were used as an in vivo marine fish species model and were fed a diet containing a synthetic antioxidant, ethoxyquin, and/or a natural antioxidant, grape seed extract, for 8 weeks. The YTK fillet was found to bioaccumulate 2.5-fold more DHA than the level supplied in the diet. However, natural fish variation resulted in substantial variation in the proportion of DHA in the fillet. Interestingly, there was a significant decrease in the proportion of DHA in the fillet after storage at 4°C for 4 days, regardless of ethoxyquin or grape seed extract antioxidant protection. Southern bluefin tuna (SBT; Thunnus maccoyii) are a large and economically valuable marine aquaculture species in South Australia. Whole SBT are essentially unobtainable for research as their supply is limited due to a strict wild-catch quota system and the inability to routinely breed them in captivity. To elucidate the enzymatic regulation of the SBT LCPUFA synthesis pathway, the Saccharomyces cerevisiae expression system was used to characterise the Elovl5 and Ä6desaturase genes from SBT liver tissue. The SBT Elovl5 and Ä6desaturase cDNAs encoded predicted proteins which had the main structural characteristic features of microsomal fatty acyl elongases and desaturases, respectively, from mammals and other fish. The Elovl5 enzyme was very efficient at elongating C18 and C20 PUFA substrates, with higher activity towards the n-3 substrates than the n-6 substrates. The Ä6desaturase enzyme activity appeared to be low because desaturation products were not detected when the cultures were supplemented with various n-3 and n-6 PUFA. However, Ä6desaturase protein expression in the S. cerevisiae system was also low, thus making it difficult to determine the substrate specificity of the Ä6desaturase. This thesis went further to show that at least one fatty acyl elongase gene is expressed in a range of SBT tissues, while expression of Ä6desaturase appears to be limited. The FHM (fathead minnow; Pimephales promelas) and CHSE-214 (Chinook salmon; Oncorhynchus tshawytscha) epithelial cell lines were used as in vitro systems to examine the LCPUFA synthesis capabilities of freshwater and anadromous fish species, respectively. The fish cell lines were supplemented with n-3 and n-6 PUFA to investigate if the LCPUFA synthesis pathway in the cell lines could be used as a model for fish in vivo. This thesis confirmed that the CHSE-214 cells had functional Ä6desaturase, Elovl5 and Elovl2 enzymes, consistent with previous data. In contrast, the FHM cell line displayed the ability to elongate PUFA substrates but did not efficiently desaturate them. The low Ä6desaturase activity in the FHM cells lead to the investigation of the expression of Ä6desaturase and Elovl5 genes in the FHM cells following n-3 PUFA supplementation. Approximately the same level of up-regulation was seen, regardless of the n-3 PUFA. This thesis highlights the different LCPUFA synthesis pathway capabilities in freshwater, anadromous and marine fish species. These findings will help define dietary approaches to maintaining or enhancing the synthesis of LCPUFA in aquaculture fish species.
Keywords: long chain polyunsaturated fatty acids,fatty acyl elongase,fatty acyl desaturase,aquaculture,fish cell lines,antioxidant,grape seed extract
Subject: Biological sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Dr Kathryn Schuller