Final Report Summary - LONGFA (Biosynthesis of very long-chain fatty acids in fish: Molecular and biochemical basis and implications in aquaculture) Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential compounds that play pivotal roles in numerous metabolic and physiological processes ensuring normal cellular function. In many ways the molecular control and basis of LC-PUFA synthesis in vertebrates is better understood in fish than in humans. This interest has driven by the crucial role that fish play as the primary source of the health-promoting n-3 LC-PUFA including eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) in the human food basket. Despite the enormous attention devoted to LC-PUFAs (C20-22), the so-called “very long-chain polyunsaturated fatty acids” (VLC-PUFAs), including compounds with carbon chains longer than C24, have been barely investigated in fish.Preliminary data suggested that in fish, two types of elongases of very long-chain fatty acid (Elovl), namely Elovl2 and Elovl4, are key enzymes in the biosynthesis of VLC-PUFAs, with Elovl2 producing VLC-PUFAs up to C26, whereas Elovl4 is involved in the biosynthesis of longer VLC-PUFAs including polyenes up to C36. The overarching aim of LONGFA project was to investigate the biosynthesis of very long-chain polyunsaturated fatty acids (VLC-PUFAs) in finfish, using the marine teleost Sparus aurata as model species. The project combined a series of fundamental objectives aiming at molecular and functional characterisation of Elovl proteins involved in the VLC-PUFA biosynthetic pathway, together with more applied objectives aiming at the effects that aquafeeds with suboptimal contents of potential VLC-PUFA precursors have for fast-growing stages of fish, where high requirements of VLC-PUFA are expected to occur.Over the course of the LONGFA project, we have identified two distinct Elovl4-like elongases from gilthead seabream, named as Elovl4a and Elovl4b according their zebrafish homologues. Their functional analysis revealed that both enzymes efficiently elongated saturated fatty acids up to 34:0. Both isoforms, particularly Elovl4b, elongated polyunsaturated fatty acid (PUFA) in an efficient way, producing unusually long PUFA like 34:5n-3 and 34:4n-6. The temporal expression patterns of gilthead seabream elovl4 isoforms was examined. We confirmed that elovl4a and elovl4b are expressed throughout the entire embryogenesis of S. aurata, indicating that active VLC-PUFA biosynthesis is required during early development. In adulthood, both elovl4a and elovl4b are widely distributed in all tissues investigated, with brain and eye confirmed to show the highest expression signals. These results confirmed our initial hypothesis by which neuronal tissues (brain and eye) were postulated as key metabolic sites for the biosynthesis of VLC-PUFAs. We further examined the presence of VLC-PUFAs in brain and eye from wild-caught S. aurata individuals. We were able to identify VLC-PUFAs in some specific phospholipid types of some particular tissues. While further identification is still in progress, the fatty acid profiles of retinal phosphatidylcholine (PC) confirmed the presence of VLC-PUFAs with chain-lengths beyond C32 and five unsaturations (double bonds). As far as we are concerned, this is the only report of the existence of VLC-PUFAs in fish since these compounds were discovered in late 80s and described to exist in several vertebrates including two species of fish.The increased expression signals of elovl4 in retina and brain of S. aurata highlight the importance that the study of VLC-PUFAs and their biosynthesis have in farmed fish in which altered visual acuity (critical in visual predators such as most cultured fish species) and disruptions of brain functioning can affect growth performance and eventually the economical profit of the farm. This is particularly true for early life-cycle stages of development, when accretion of these compounds in forming tissues occurs. Thus LONGFA aimed to investigate the effect of potentially deficient diets have on normal development and VLC-PUFA biosynthetic pathway of fast-growing stages of fish. A 4-month feeding trial with S. aurata juveniles (~ 1-2 g weigh) fed on diets with varying FO contents is currently running at the facilities of the Instituto de Acuicultura Torre de la Sal (IATS-CSIC), the host institution. While the trial will be finished by the end of November 2013, tissue samples and biometrics data have been collected monthly. We will be able to study the regulation of the LC- and VLC-PUFA biosynthetic pathways through expression analysis (qPCR) of key desaturase (fads2) and elongase (elovl5, elovl4a and elovl4b) genes. Moreover, the fatty acid composition will be also determined, with special focus on the identification of VLC-PUFAs in key tissues such as retina and brain.Along with the results directly derived from LONGFA project itself, further studies in connection to LC-PUFA biosynthetic pathways in aquatic organisms have been also approached. The results of some of those further studies have been published in scientific journals or presented in scientific conferences. These further studies allowed the fellow to fully integrate in the projects of his host group at IATS-CSIC and, more importantly, to establish both national and international collaborations. Clearly the reintegration of Dr. Monroig at the host institution has resulted in a mutual benefit for both the fellow and the host group.
