The primary aim of this study was to characterise Northern pike Elovl5, a critical enzyme of LC-PUFA biosynthesis in vertebrates. The precise reasons for undertaking the work were ultimately to gain understanding of the evolutionary and ecological adaptations of salmonids. Phylogenetic evidence indicates that esocids are the nearest living relatives of salmonids, having diverged at some point prior to a WGD event in the common ancestor of all salmonids between 25 and 100 mya [1, 2]. As WGD has been widely suggested as a major enabling event in evolutionary innovation , comparison of single preduplicated genes in pike with their duplicated paralogs in Atlantic salmon has the potential to shed light on evolutionary mechanisms and adaptation in salmonids. The genes of the LC-PUFA biosynthetic pathway are interesting candidates for studies of this type because both genetic and biochemical evidence suggests that salmonids have a higher LC-PUFA biosynthetic capacity than many other fish species [11, 20]. Atlantic salmon have duplicated genes for both desaturases and elongases of fatty acids and, in the case of desaturases, duplicates appear to have diverged and neo- or subfunctionalised to provide enzymatic activities for the entire LC-PUFA pathway [26, 27]. In contrast, other fish species, particularly marine carnivorous species, are incapable of endogenous production of significant amounts of LC-PUFA because they lack critical genes of the biosynthetic pathway [17, 25, 35]. It has been suggested that this enhancement of LC-PUFA capacity in salmonids has been a factor in their success in colonising relatively nutrient poor freshwater environments . Salmonids can thrive, and are often the only fish group, in flowing bodies of water, which are reliant on allochthonous terrestrial inputs as a major source of energy. Terrestrial inputs whether directly from leaf litter, or from insect species are poor sources of LC-PUFA, especially DHA, in contrast with food sources in marine or eutrophic freshwater environments which are underpinned by blooms of phytoplankton rich in LC-PUFA [17, 20, 35, 36]. Despite the fact that the tissues of freshwater and marine fish are generally rich in C20 and C22 LC-PUFA, the strategies they utilise to fulfil such requirements vary depending on the species and the dietary input. Northern pike is a strictly freshwater species, whose distribution overlaps that of salmonids, and which shares a relatively recent common ancestor with salmonids. However, pike differ from salmonids in exhibiting a far more piscivorous feeding behaviour. Hence, our particular interest in LC-PUFA biosynthetic genes in this species in comparison to those in Atlantic salmon.
Genetic linkage analyses established that the Atlantic salmon elovl5 duplicates are located on different linkage groups: elovl5a on LG 33, and elovl5b on LG 5. Cytogenetic mapping using fluorescence in situ hybridisation has assigned salmon LG 33 and LG 5 to chromosomes 28 and 13, respectively [37, 38], both of which are acrocentric. While our data clearly show that the two elovl5 loci are not physically linked, in a recent analysis  the Atlantic salmon chromosomes ssa28 and ssa13 were not homeologous and it is therefore not possible to conclude that the salmon elovl5 paralogous genes are the result of a WGD. It is also possible that this duplication is unique to Atlantic salmon, since no clear evidence of duplicated elovl5 genes in other salmonids exist in the current sequence databases. However, compared to all other salmonids which have c. 100 chromosome arms, Atlantic salmon is unique in possessing only 72–74 chromosome arms, believed to be the result of species-specific tandem fusions and other rearrangements . Furthermore, linkage maps show  that salmon chromosome ssa13 has homeologous regions on at least three other salmon chromosomes, and shares syntenic blocks with at least four separate chromosomes from the diploid stickleback (Gasterosteus aculeatus). Thus, given the complexity of the Atlantic salmon genome, it would be premature to reject a WGD origin for the two salmon elovl5 loci.
Phylogenetic analysis confirmed the basal nature of Northern pike within the protacanthopterigyans. Analysis of the rates of nucleotide substitution in the Atlantic salmon Elovl5 paralogous genes indicated that they are under an evolutionary regime of purifying selective pressure (ω < 1), and are currently evolving at comparable evolutionary rates at the protein level, thus supporting the idea that both duplicates are physiologically required, and have the same biochemical activity as the pike Elovl5. In a larger phylogenetic study,  performed pairwise dN/dS analyses on 408 sets of duplicated salmon genes using a preduplicated set of Northern pike orthologs as outgroups, and similarly concluded that salmon paralogs were predominantly exposed to purifying selection, although some loci may be showing a relaxation of selective pressure suggesting that evolution was acting asymmetrically on some paralogs due to reduced constraints. A closer inspection of the dN/dS along the coding sequences of vertebrate elovl5 suggested the accommodation of a major catalytic site where stronger functional constraints seemed to have acted against the retention of nonsynonymous mutations that would compromise the performance of the enzyme, or impair its activity [40, 41].
Here we also tested for functional similarity of pike Elovl5 to salmon paralogs by heterologous expression of the pike enzyme in yeast. Supporting the phylogenetic results, the activity of the pike Elovl5 was indistinguishable from previous assays of the salmon paralogs . Pike Elovl5 was able to lengthen PUFA substrates with chain lengths from C18 to C22. The 18:4n-3, 18:3n-6 and C20 specificity of pike Elovl5 and both salmon Elovl5 paralogs was very similar, and similar to that in other vertebrates . Only very low, residual activity for the production of 24:5n-3 was detected in yeast transformed with the pike elongase when incubated with 22:5n-3, as previously observed in other species, including salmonids [25, 32, 42]. As 24:5n-3 is an important intermediate in most vertebrates for the biosynthesis of DHA, and in salmonids C22 to C24 activity has been demonstrated in Elovl2 and Elovl4 [25, 28], it would be interesting to look for and study these elongase genes in pike.
Until recently the production of LC-PUFA from C18 PUFA was thought to proceed via an alternating desaturation/elongation cycle with the initial step being Δ6 desaturation. However, recent evidence suggests that an alternative pathway is also possible, with the initial step being C18 to C20 elongation, based upon the ability of Δ6 desaturases to also catalyse Δ8 desaturation. For such an alternative loop to exist, elongase activity on LA and ALA is required, and this has been demonstrated in other species [42–44] and, according to results presented here, is also shown by pike Elovl5. Although not yet demonstrated in salmon, we would expect that, given the Δ8 activity of salmon Δ6 desaturases , salmon Elovl5 enzymes would also possess this activity.
Although the phylogenetic and functional analyses point towards the maintenance of ancestral activities, the expression profiles indicate functional partitioning in the salmon elovl5 paralogs. Salmon Elovl5a and Elovl5b have different tissue expression profiles, with Elovl5a being expressed at higher level in intestine and Elovl5b in liver . In addition, the nutritional regulation of mRNA transcription of these genes differs in tissues from fish fed diets containing low levels LC-PUFA . The tissue distribution of pike Elovl5 transcripts showed that the highest expression across the tissues tested was in brain, whereas most other tissues, including liver and intestine, showed very low expression in comparison. In contrast, salmon Elovl5 transcripts were predominantly expressed in liver and intestine, with much lower expression in brain, and even less in other tissues . The pattern of pike Elovl5 tissue expression closely resembles the pattern of LC-PUFA biosynthetic gene expression in carnivorous marine fish, where expression and activity is low in liver and intestine, but high in the brain . Brain, as with all neural tissues, has a high LC-PUFA level [17, 46] but, as yet, mechanisms for the biosynthesis, or the transport of fatty acids to the brain are not fully understood . Mammalian brain is only capable of biosynthesising a restricted set of fatty acids  and it is clear that fatty acid uptake in the brain is different to uptake in most other tissues, probably due the requirement for passage across the blood–brain barrier. Radiolabeled PUFA injected intraperitoneally in Northern pike could be detected in high concentrations throughout the body, with the exception of brain, which consistently contained the lowest amounts of injected PUFA . Currently, most studies support an energy-dependant mechanism that facilitates and regulates fatty acid uptake influenced by chain length and degree of unsaturation [49, 50]. The gene expression results from pike and other carnivorous fish suggest that brain may have endogenous biosynthetic machinery for LC-PUFA to supply the high requirements of this tissue, whereas low expression in liver and intestine indicate a reduced requirement for LC-PUFA, or at least DHA, in these tissues. Although low levels of LC-PUFA biosynthesis have been detected in isolated pike hepatocytes , experiments in vivo failed to find evidence of significant conversion in liver . In salmon, although Elovl5 and other LC-PUFA biosynthetic genes are expressed in brain, the highest expression levels are in liver and intestine, the main tissues responsible for dietary fatty acid uptake, biosynthesis and distribution [17, 25]. Salmonid liver has a comparatively high capacity for biosynthesising LC-PUFA such as EPA and DHA , whereas marine carnivorous fish, such as sea bass (Dicentrarchus labrax) have negligible capacity depending on dietary LC-PUFA .
This above discussion should be qualified by noting that studies on various salmonids and other freshwater species fed with artificial diets with varying LC-PUFA contents and compositions have shown that hepatic desaturase and elongase enzymes exhibited higher expression when the amount of dietary LC-PUFA decreased and LA and/or ALA increased [11, 20, 25, 51]. Although, it is possible that LC-PUFA biosynthetic enzymes in liver are also under nutritional regulation in pike, the fish in the present study were obtained from wild stock and thus essentially piscivorous and so would be equivalent to salmon fed diets containing fish oil (i.e. high in LC-PUFA) .