Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris: Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase and an Elongation of Very Long-Chain Fatty Acid 4 Protein

Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C18 chain lengths. Scd was unable to desaturate 20:1n-15 (∆520:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1n-9 (∆1120:1) to ∆5,1120:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5n-3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C24) PUFAs.


Introduction
Cephalopods have been regarded as promising candidates for the diversification of marine aquaculture due to their great commercial interest [1].Despite significant progress made over the last decade, culture of cephalopod species with pelagic paralarval stages like the common octopus Octopus vulgaris is still challenging due to the massive mortalities occurring upon the settlement phase [2].The specific factors causing such mortalities of paralarvae remain unclear, although it has become increasingly obvious that nutritional issues associated with inadequate supply of essential nutrients such as lipids are crucial to ensure normal growth and development of O. vulgaris paralarvae and ultimately improve their viability [3].
Previous investigations postulated that polyunsaturated fatty acids (PUFAs) are essential nutrients for the common octopus [4,5].However, the specific PUFAs that satisfy the physiological requirements were not determined, partly due to the difficulties in running nutritional trials on octopus paralarvae.In order to provide insights to the endogenous capability for PUFA biosynthesis in O. vulgaris, we have recently conducted a series of studies aiming to identify and characterise the function of genes encoding enzymes that mediate the conversions in the PUFA biosynthetic pathways.First, we identified a fatty acyl desaturase (Fad) cDNA sequence with homology to the vertebrate Fads family [6], enzymes that participate in long-chain (C 20-24 ) PUFA (LC-PUFA) biosynthetic pathways [7][8][9].The expression of the common octopus Fad in yeast demonstrated that the enzyme was a D5 desaturase (D5 Fad) with high efficiency towards both saturated and polyunsaturated fatty acid (FA) substrates [6].Thus, the O. vulgaris D5 Fad was able to desaturate the yeast endogenous saturated FAs 16:0 and 18:0 to the corresponding monoenes 16:1n-11 ( D5 16:1) and 18:1n-13 ( D5 18:1), respectively.Furthermore, the O. vulgaris D5 Fad efficiently desaturated the PUFA 20:4n-3 and 20:3n-6 to the D5 desaturation products eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), respectively (Figure 1).
A second study provided further evidence of the existence of an active PUFA biosynthetic system in the common octopus [10].Thus, a cDNA encoding a protein with high homology to an elongation of very long-chain fatty acids (Elovl) protein was isolated [10].Phylogenetic analysis comparing the amino acid (aa) sequence of the O. vulgaris Elovl with other elongases from molluscs and vertebrates clearly showed that the common octopus Elovl, as well as other putative elongases from molluscs, was grouped as a basal cluster of the vertebrate Elovl2 and Elovl5 families [10].Consequently, such an elongase has been termed "Elovl5/2" [11] or "Elovl2/5" [12,13].Regarding its function, the common octopus Elovl2/5 exhibited substrate specificities resembling those of vertebrate Elovl5 but not Elovl2, as it efficiently elongated C 18-20 PUFAs [10] but had no activity towards C 22 substrates.This was hypothesised as one of the reasons accounting for the inability of cephalopods to biosynthesise docosahexaenoic acid (DHA; 22:6n-3) (Figure 1) [10,11] through the so-called "Sprecher pathway" requiring the production of 24:5n-3 as an intermediate in the pathway [14].Among alternative Elovl-like enzymes with a role in the biosynthesis of LC-PUFAs such as DHA in the common octopus, the Elovl4 elongase is an interesting candidate, as studies on teleost orthologues have shown that Elovl4 can efficiently catalyse the elongation of 22:5n-3 to 24:5n-3 [15][16][17][18][19], and recent studies have further demonstrated a similar elongation ability in molluscs [12].
The above studies on O. vulgaris [6,10], as well as those on homologous genes from the common cuttlefish Sepia officinalis [20], have enabled us to predict the biosynthetic pathways of PUFAs in cephalopods (Figure 1).Beyond the biosynthesis of standard PUFAs, i.e., FAs whose double bonds are always separated by a methylene group (-CH 2 -) [9], one can predict that some pathways involving the D5 Fad and Elovl2/5 lead to the production of so-called "non-methylene-interrupted FAs" (NMI FAs), a particular type of PUFA that had been previously reported in other molluscan classes (bivalves, gastropods), as well in sponges, echinoderms and other phyla [21][22][23].Analyses performed in wild-caught specimens of O. vulgaris confirmed that the polar lipid fractions of nephridium, male gonad, eye and caecum contained NMI FAs identified as D5,11 20:2, D7,13 20:2, D5,11,14 20:3 and D7,13 22:2 [10].From the unsaturation pattern of these compounds, it became clear that, in addition to D5 Fad, a further desaturase with D9 activity was likely involved in the NMI FA biosynthetic pathways accounting for the D5,9 unsaturation patterns typically found among these compounds [22,23].The stearoyl-CoA desaturase (Scd), an enzyme that is expressed in virtually all living organisms [24], has D9 desaturation capability and thus appears to play a role in NMI FA biosynthesis [7].
Our overall aim is to characterise the biosynthetic pathways of PUFAs including NMI FAs in cephalopods.Using the common octopus O. vulgaris as model species, we herein isolated two cDNAs, namely Scd and Elovl4 sequences, and characterised their functions by heterologous expression in yeast.In order to establish the mechanisms accounting for biosynthesis of D5,9 dienes (NMI FA) we further investigated the roles that the herein characterised Scd and the previously reported D5 Fad [6] play within these pathways.further investigated the roles that the herein characterised Scd and the previously reported Δ5 Fad [6] play within these pathways.

Octopus vulgaris Scd Sequence
The O. vulgaris Scd-like cDNA consisted of a 981-bp open reading frame (ORF) encoding a putative protein of 326 amino acids (aa) with a predicted molecular weight of 37.9 kDa.Its sequence was deposited in the GenBank database with the accession number JX310655.In common with other Scd proteins, the O. vulgaris putative Scd possessed three histidine boxes (HXXXH, HXXHH and HXXHH), four membrane-spanning regions rich in hydrophobic aa, and lacked the cytochrome b5 domain characteristic of Fads (Figure 2).

Octopus vulgaris Scd Sequence
The O. vulgaris Scd-like cDNA consisted of a 981-bp open reading frame (ORF) encoding a putative protein of 326 amino acids (aa) with a predicted molecular weight of 37.9 kDa.Its sequence was deposited in the GenBank database with the accession number JX310655.In common with other Scd proteins, the O. vulgaris putative Scd possessed three histidine boxes (HXXXH, HXXHH and HXXHH), four membrane-spanning regions rich in hydrophobic aa, and lacked the cytochrome b5 domain characteristic of Fads (Figure 2).

Octopus Elovl4-Like Sequence
The Elovl4-like cDNA consisted of an ORF of 930 bp whose deduced protein had 309 aa with a predicted molecular weight of 37.9 kDa (deposited in GenBank database with accession number KJ590963).From analogy to vertebrate orthologues [15][16][17][18], five putative transmembrane domains containing hydrophobic aa stretches can be predicted (Figure 3).The common octopus putative Elovl4 contained the histidine dideoxy-binding motif HXXHH, and the putative endoplasmic reticulum (ER) retrieval signal with a histidine (H) and lysine (K) residues at the carboxyl terminus, HXKXX (Figure 3) [26].

Octopus Elovl4-Like Sequence
The Elovl4-like cDNA consisted of an ORF of 930 bp whose deduced protein had 309 aa with a predicted molecular weight of 37.9 kDa (deposited in GenBank database with accession number KJ590963).From analogy to vertebrate orthologues [15][16][17][18], five putative transmembrane domains containing hydrophobic aa stretches can be predicted (Figure 3).The common octopus putative Elovl4 contained the histidine dideoxy-binding motif HXXHH, and the putative endoplasmic reticulum (ER) retrieval signal with a histidine (H) and lysine (K) residues at the carboxyl terminus, HXKXX (Figure 3) [26].

Functional Characterisation of the Octopus Scd
Yeast Saccharomyces cerevisiae cells lacking the OLE1 gene are unable to synthesise Δ9monounsaturated FAs including palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), which are essential for growth [28].To address whether the O. vulgaris Scd was able to complement yeast OLE1 function, an S. cerevisiae ole1∆ mutant strain (L8-14C) was transformed with a plasmid expressing the octopus Scd (p416OLE1-Scd) under the control of yeast OLE1 promoter.The ole1∆ mutant yeast were also transformed with a plasmid expressing the S. cerevisiae OLE1 (∆9 desaturase) under the control of its own promoter (p416OLE1-OLE1) as a positive control, and with empty vector (pRS416) as a negative control.All three yeast transformants were able to grow in media supplemented with at least one supplemented FA (16:1n-7 and/or 18:1n-9) (Figure 4a-c).However, only yeast cells expressing either the S. cerevisiae OLE1 or the O. vulgaris Scd grew on medium that was not supplemented with monounsaturated FAs (Figure 4d).These results indicated that the common octopus Scd complemented the function of the yeast OLE1 desaturase.

Functional Characterisation of the Octopus Scd
Yeast Saccharomyces cerevisiae cells lacking the OLE1 gene are unable to synthesise D9-monounsaturated FAs including palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), which are essential for growth [28].To address whether the O. vulgaris Scd was able to complement yeast OLE1 function, an S. cerevisiae ole1D mutant strain (L8-14C) was transformed with a plasmid expressing the octopus Scd (p416OLE1-Scd) under the control of yeast OLE1 promoter.The ole1D mutant yeast were also transformed with a plasmid expressing the S. cerevisiae OLE1 (D9 desaturase) under the control of its own promoter (p416OLE1-OLE1) as a positive control, and with empty vector (pRS416) as a negative control.All three yeast transformants were able to grow in media supplemented with at least one supplemented FA (16:1n-7 and/or 18:1n-9) (Figure 4a-c).However, only yeast cells expressing either the S. cerevisiae OLE1 or the O. vulgaris Scd grew on medium that was not supplemented with monounsaturated FAs (Figure 4d).These results indicated that the common octopus Scd complemented the function of the yeast OLE1 desaturase.

Functional Characterisation of the Octopus Scd
Yeast Saccharomyces cerevisiae cells lacking the OLE1 gene are unable to synthesise Δ9monounsaturated FAs including palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), which are essential for growth [28].To address whether the O. vulgaris Scd was able to complement yeast OLE1 function, an S. cerevisiae ole1∆ mutant strain (L8-14C) was transformed with a plasmid expressing the octopus Scd (p416OLE1-Scd) under the control of yeast OLE1 promoter.The ole1∆ mutant yeast were also transformed with a plasmid expressing the S. cerevisiae OLE1 (∆9 desaturase) under the control of its own promoter (p416OLE1-OLE1) as a positive control, and with empty vector (pRS416) as a negative control.All three yeast transformants were able to grow in media supplemented with at least one supplemented FA (16:1n-7 and/or 18:1n-9) (Figure 4a-c).However, only yeast cells expressing either the S. cerevisiae OLE1 or the O. vulgaris Scd grew on medium that was not supplemented with monounsaturated FAs (Figure 4d).These results indicated that the common octopus Scd complemented the function of the yeast OLE1 desaturase.To analyse in more detail the complemention of yeast ole1D mutants by the O. vulgaris Scd, we determined the FA composition of ole1D yeast cells transformed with either p416OLE1-OLE1 (i.e., expressing the S. cerevisiae OLE1) or p416OLE1-Scd (i.e., expressing the O. vulgaris Scd), and grown in liquid medium with no exogenously supplemented FAs.Yeast expressing the O. vulgaris Scd grew notably slower compared to controls and formed clumps as previously described [29].Moreover, the p416OLE1-Scd yeast contained significantly (p  0.05) less 16:1n-7 and more 16:0 than control yeast expressing the S. cerevisiae OLE1, suggesting a low affinity of the O. vulgaris Scd towards 16:0.On the contrary, contents of 18:1n-9 showed no statistical differences between yeast cells expressing Scd or OLE1, indicative of the octopus Scd participating in the biosynthesis of 18:1n-9 similar to S. cerevisiae Ole1p.The distinctive substrate specificities of the O. vulgaris Scd were further emphasised by the higher 18:1n-9/16:1n-7 ratio of yeast complemented with the O. vulgaris Scd compared to that of OLE1 control yeast (Table 1).
The substrate specificities of the O. vulgaris Scd were further investigated through an overexpression assay using the S. cerevisiae strain InvSc1 (Invitrogen, Paisley, UK), possessing the endogenous Ole1p.Comparison of the FA profiles between the control yeast (i.e., transformed with the empty pYES2) and yeast expressing the O. vulgaris Scd (i.e., transformed with pYES2-Scd) confirmed a role of the common octopus Scd in the biosynthesis of monounsaturated FAs (Table 2).Thus, significant (p  0.05) increases in the contents of 18:1n-9 (oleic acid) in pYES2-Scd yeast compared to control yeast were observed.Parallel decreased levels of the saturated FA precursor 18:0 were detected in pYES2-Scd yeast (p  0.05).Additionally, other monoenes corresponding to 20:1n-11 and 22:1n-13 were detected in total lipids of InvSc1 yeast expressing the O. vulgaris Scd.These results confirmed that the O. vulgaris Scd is a D9 desaturase with activity towards saturated FAs with chain-lengths C 18 .In contrast, shorter FAs (C 16 ) did not appear to be adequate substrates for the octopus Scd and, for instance, the content of 16:1n-7 in yeast expressing the common octopus Scd was significantly lower than that of control yeast (p  0.05) (Table 2).In order to establish the biosynthetic pathways of NMI FAs with D5,9 unsaturation patterns (or their derivatives such as D5,11) found in the common octopus lipids [10], InvSc1 yeast transformed with pYES2-Scd were grown in the presence of the monoene 20:1n-15 ( D5 20:1), while InvSc1 yeast expressing the D5 Fad (i.e., transformed with pYES2-Fad) [6] were grown in the presence of 20:1n-9 ( D11 20:1) (Figure 5).Our results showed that the O. vulgaris Scd was unable to desaturate the substrate D5 20:1 (20:1n-15) to D5,9 20:2 (Figure 5a), although yeast transformed with pYES2-Fad were able to produce D5,11 20:2 (2.7% ± 0.2% conversion), thus confirming activity as D5 desaturase on D11 20:1) (Figure 5b).

Discussion
Fish and seafood are the primary sources of omega-3 long-chain (C 20-24 ) PUFAs for humans [31] and this partly explains the considerable interest in elucidating the PUFA biosynthetic pathways in aquatic and marine species, particularly in farmed fish for which current trends in feed formulation are impacting the nutritional quality for human consumers [32].Previous investigations on O. vulgaris [6,10,33] and S. officinalis [20] revealed that cephalopods possess active desaturase and elongase enzymes involved in the biosynthesis of PUFAs including NMI FAs.Using the common octopus O. vulgaris as model species, the present study aimed to expand our knowledge of roles that further desaturases and elongases could have on PUFA biosynthesis in cephalopods.Consequently, we characterised a stearoyl-CoA desaturase (Scd) with a putative role in the biosynthesis of NMI FAs, and an Elovl4 elongase that, in addition to its participation in the biosynthesis of VLC-PUFAs [15][16][17][18][19]34], could potentially catalyse the elongation of 22:5n-3 to 24:5n-3 required for DHA biosynthesis through the Sprecher pathway [14].
The Scd, present in virtually all living organisms [24], is an enzyme with D9 desaturase activity.Consistently, the newly-cloned O. vulgaris Scd cDNA was confirmed to encode a D9 desaturase that was able to operate on a range of saturated FA substrates with different chain lengths, particularly C 18 .These functions are similar to those described for two homologous sequences found in the nematode C. elegans, namely FAT-6 and FAT-7, which can efficiently desaturate 18:0 to 18:1n-9 but have lower activity towards 16:0 [25].Interestingly, FAT-5, another Scd-like sequence existing in C. elegans, efficiently desaturated 16:0 to 16:1n-7 but had nearly undetectable activity on 18:0, and it is thus regarded as a palmitoyl-CoA-specific desaturase [25].The functional characterisation of the O. vulgaris Scd and its particularly high substrate affinity towards 18:0 is consistent with 18:1n-9 typically appearing several folds above the levels of shorter monoenes such as 16:1n-7 in lipids across the Mollusca phylum [21].Beyond its role in biosynthesis of monounsaturated FA, occurrence of NMI FAs with D5,9 unsaturation patterns in lipids from molluscs [22] suggested a role of Scd in the biosynthesis of this particular type of PUFA.Our results revealed that the O. vulgaris Scd was not able to introduce a second double bond on a D5 monoene such as 20:1n-15 ( D5 20:1).While the possibility that cephalopod Scd can introduce D9 desaturations on other monoenes cannot be ruled out, these results strongly suggest that the role of Scd in the biosynthesis of NMI FAs with D5,9 unsaturation patterns is limited to the insertion of the first unsaturation at D9 position on saturated FAs.Indeed, the O. vulgaris D5 Fad, previously characterised as D5 desaturase [6], was able to introduce a D5 unsaturation on 20:1n-9 ( D11 20:1) producing the NMI FA D5,11 20:2.Overall, these results allow us to hypothesise that the insertion of double bonds required for NMI FA biosynthesis is initiated with a D9 desaturation by Scd and a subsequent D5 desaturation catalysed by D5 Fad.Compared to other molluscan classes, the occurrence of NMI FAs in cephalopods appears to be rather limited and hence the contribution of endogenous production of NMI FAs vs. dietary input is difficult to establish.Nevertheless, the mechanism of D5,11 20:2 biosynthesis postulated herein aligns well with the occurrence of D5,11 20:2 in the polar lipids of nephridia of adult O. vulgaris [10], suggesting that NMI FA biosynthesis is possible in cephalopods and likely other molluscs.
In addition to desaturases, certain Elovl enzymes play crucial roles in the biosynthesis of essential PUFAs such as EPA, ARA and DHA [9,11,26].Whereas the previously characterised cephalopod Elovl2/5 demonstrated an ability to efficiently elongate C 18 and C 20 PUFA substrates [6,20], lack of elongase capability towards 22:5n-3 was hypothesised as a major limiting factor for DHA biosynthesis through the Sprecher pathway [6,20].Functional characterisation of the O. vulgaris Elovl4 demonstrated the ability of this enzyme to contribute potentially to DHA biosynthesis by producing 24:5n-3, which is the substrate for D6 desaturation and chain-shortening to produce DHA in the Sprecher pathway [14].Such an ability of Elovl4 has been reported in fish orthologues [15][16][17][18][19].Despite the elongation capability of O. vulgaris herein revealed, our overall observations on gene repertoire and function [6,10,20,33] strongly suggest that DHA is still an essential FA for cephalopods.The limited ability to biosynthesise DHA is related, rather than to an issue with elongase activity, to the apparent absence of key desaturation abilities (D6 or D4) required for DHA biosynthesis according to the two aerobic pathways known in vertebrates (Figure 1).In addition to the abovementioned D6 desaturation required in the Sprecher pathway [14], an alternative pathway described in some teleost fish involves a D4 desaturation from 22:5n-3 to produce DHA directly (Figure 1) [9].Molecular evidence provided by the recently published Octopus bimaculoides genome project [35] suggests that cephalopods possess one single Fad-like desaturase in their genome and this is likely to be a D5 desaturase since all the Fad-like desaturase functionally characterised not only from cephalopods such as O. vulgaris and S. officinalis [6,10] but also from bivalves [36] and gastropods [37], have D5 desaturase activities.In non-cephalopod molluscs, further Fad-like desaturases exist [38] but, with the exception of the D8 desaturase found in the scallop Chlamys nobilis [39], their functions have not been determined and therefore it is yet not possible to predict whether D6 and/or D4 desaturase activities exist to enable DHA biosynthesis in those species.
The O. vulgaris Elovl4 is also involved in the biosynthesis of VLC-PUFAs as it was able to elongate exogenously supplemented PUFAs ranging from C 18-24 to PUFA products with chain lengths of C 26 .Such conversions are largely consistent with those previously exhibited by human [34] and fish [15][16][17][18][19] Elovl4 proteins.Surprisingly, no elongation products beyond C 24 were reported in the functional characterisation of the bivalve C. nobilis Elovl4 [39].The elongation activities of the O. vulgaris Elovl4 estimated in the yeast expression system strongly suggested that this enzyme is particularly efficient towards C 26-30 substrates, for which the highest % conversions were observed.Similarly, the human ELOVL4 had PUFAs of C 26 as preferred substrates for elongation, although the human ELOVL4 was able to produce up to C 38 PUFA elongation products in mammalian cell lines [34].Due to technical challenges in their analysis and relatively low abundance, the functions of VLC-PUFAs are not fully understood [40].Nevertheless, the structural features of VLC-PUFAs combining those from saturated FAs at one end and those from PUFAs at the other allow unique membrane lipid conformations in photoreceptors and spermatozoa, thus suggesting important roles of VLC-PUFAs in vision and reproduction of vertebrates [40][41][42][43].Investigations of VLC-PUFAs in cephalopods and, therefore, the herein reported activities of the O. vulgaris Elovl4 cannot be correlated with the presence of VLC-PUFAs in vivo.Interestingly, some very long-chain NMI FAs have been reported in nudibranchs [44,45] but, unfortunately, the potential role of Elovl4 in the biosynthesis of such compounds was not determined.Interestingly, unpublished data on tissue distribution analysis of the common octopus Elovl4 mRNA indicated that gonads and, to a lesser extent eye, were also major sites for VLC-PUFA biosynthesis in cephalopods.
In conclusion, the present study demonstrated that the common octopus O. vulgaris possesses an Scd with high affinity for saturated FA substrates with chain lengths of 18 carbons or longer.Its inability to introduce a double bond into D5 monoene strongly suggested that the role of the Scd in NMI FA biosynthesis was restricted to the introduction of the first double bond at the D9 position, whereas the O. vulgaris D5 Fad can introduce the second unsaturation at the D5 position according to the herein confirmed ability to convert 20:1n-9 to D5,11 20:2, an NMI FA previously reported in nephridia of O. vulgaris adult specimens.Beyond desaturases, we could also demonstrate that O. vulgaris possesses an Elovl4 responsible for the biosynthesis of VLC-PUFAs.

Tissue Samples
Tissue samples including brain, nerve, muscle, heart, hepatopancreas, gill, caecum, eye, nephridium and gonads were obtained from the dissection of two (male and female) common octopus adult specimens as previously described [6,10].Briefly, two (male and female) wild O. vulgaris adults (~1.5 kg) were maintained in seawater tanks at the facilities of the Instituto de Acuicultura Torre de la Sal; before they were cold, they were anesthetised and sacrificed by direct brain puncture.After collection, samples were immediately frozen at 80 C until further analysis.Total RNA was extracted from octopus tissues using TriReagent ® (Sigma-Aldrich, Alcobendas, Spain) according to manufacturer's instructions.Two µg of total RNA tissue samples were used for synthesis of first strand cDNA using M-MLV reverse transcriptase (Promega, Southampton, UK) primed with random hexamers.
Polymerase chain reactions (PCR) were performed with the GoTaq ® Green Master Mix (Promega) and using a mixture of cDNAs from brain, nerve and hepatopancreas as template.The PCR consisted of an initial denaturing step at 95 C for 2 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 50 C for 30 s, extension at 72 C for 40 s, followed by a final extension at 72 C for 5 min.PCR products of approximately 180 bp (Scd) and 290 bp (Elovl4) were obtained and thereafter confirmed as positive by sequencing (DNA Sequencing Service, IBMCP-UPV, Valencia, Spain).Full-length cDNA of the octopus Scd and Elovl4 were completed by 5 0 and 3 0 rapid amplification of cDNA ends (RACE) PCR (FirstChoice RLM-RACE kit, Ambion, Applied Biosystems, Warrington, UK) with gene-specific primers shown in Table S1.

Sequence Analysis
The deduced aa sequences of the O. vulgaris Scd and Elovl4 ORF were compared to corresponding orthologues from other invertebrate and vertebrate species and sequence identity scores were calculated using the EMBOSS Needle Pairwise Sequence Alignment tool (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Deduced aa sequence alignments were carried out using the built-in ClustalW tool (BioEdit v7.0.9, Tom Hall, Department of Microbiology, North Carolina State University, Raleigh, NC, USA).

Functional Characterisation of Octopus Scd: Complementation Assay of the S. cerevisiae ole1D Mutant Strain L8-14C
The open reading frame (ORF) of the common octopus Scd was amplified from a mixture of cDNAs (brain, nerve and hepatopancreas) using the high fidelity Pfu Turbo DNA polymerase (Promega) and the primers OVD9VF and OVD9VR, containing restriction sites HindIII and XhoI, respectively (underlined in Table S1).PCR conditions consisted of an initial denaturing step at 95 C for 2 min, followed by 32 cycles of denaturation at 95 C for 30 s, annealing at 58 C for 30 s, extension at 72 C for 2.5 min, followed by a final extension at 72 C for 5 min.After restriction of the PCR product, the O. vulgaris Scd ORF was cloned into the yeast expression vector p416TEF (a centromeric plasmid with a URA3 selectable marker) to produce the construct p416TEF-Scd, in which octopus Scd was under the control of the yeast TEF1 promoter.Subsequently, the promoter region of the S. cerevisiae OLE1 D9-fatty acid desaturase gene amplified from yeast genomic DNA with the primers SCPromOLE1F and SCPromOLE1R containing SacI and HindIII sites, respectively, was cloned upstream of the O. vulgaris Scd, replacing the TEF1 promoter and producing the construct p416OLE1-Scd, in which octopus Scd is expressed under the control of the yeast OLE1 promoter.As a positive control, the promoter and ORF of S. cerevisiae OLE1 were amplified by PCR from yeast genomic DNA using the primers SCPromOLE1F and SCOLE1R that contained restriction sites for SacI and XhoI, respectively (Table S1).To obtain the p416OLE1-OLE1 plasmid, the OLE1 PCR fragment was cloned into p416TEF SacI and XhoI restriction sites, allowing TEF1 promoter replacement by OLE1 promoter and ORF.

Functional Characterisation of Octopus Scd: Overexpression in the S. cerevisiae Strain InvSc1
Primers containing HindIII (forward) and XhoI (reverse) restriction sites (underlined in Table S1) OVD9VF and OVD9VR (Scd) were used to amplify the ORF of the O. vulgaris Scd, using the high fidelity Pfu Turbo DNA polymerase (Promega).Further cloning into the yeast expression vector pYES2 (Invitrogen), which contains a GAL1 promoter that is inducible by galactose and has URA3 as selective marker, was achieved after ligation of restricted ORF amplicons and plasmid pYES2 to produce the construct pYES2-Scd.The recombinant plasmids pYES2-Scd or pYES2 empty (negative control) were transformed into S. cerevisiae competent cells InvSc1 (S.c.EasyComp Transformation Kit, Invitrogen).Yeast were grown in SC-ura for 3 days.
One single yeast colony containing the pYES2-Scd or pYES2 was grown overnight at 30 C in 5 mL of liquid SC-ura.Cell cultures were then used to inoculate 10 mL of fresh SC-ura for a final OD 600 of 0.4.Four replicates for each construct (pYES2-Scd or pYES2) were run.Cells were grown at 30 C for 5 h before the expression of the transgene was induced by the addition of galactose to 2% (w/v) [46].After 48 h of galactose induction, yeast samples were collected, washed and homogenised in chloroform/methanol (2:1, v/v) containing 0.01% BHT.Samples were kept at 20 C until further analysis.

Functional Characterisation of Octopus Elovl4: Expression in the S. cerevisiae Strain InvSc1
The octopus Elovl4 was functionally characterised by heterologous expression in yeast S. cerevisiae (strain InvSc1, Invitrogen).Similarly, as described above for the Scd overexpression experiment, the octopus Elovl4 ORF was amplified with primers OVE4VF and OVE4VR containing restriction sites for HindIII and XhoI, respectively (Table S1) which allowed its cloning into pYES2 to produce the construct pYES2-Elovl4.Yeast InvSc1 transformed with pYES2-Elovl4 were grown in SC-ura plates.One colony was subsequently grown in SC-ura broth to produce a bulk culture that allowed us to establish subcultures of OD 600 0.4 in Erlenmeyer flasks that contained 5 mL of SC-ura broth and were in some cases supplemented with potential PUFA substrates for fatty acyl elongases.In order to assess the ability of the octopus Elovl4 to elongate PUFA substrates, yeast transformed with pYES2-Elovl4 were grown in the presence of one of the following PUFA substrates: 18:3n-3, 18:2n-6, 18:4n-3, 18:3n-6, 20:5n-3, 20:4n-6, 22:5n-3, 22:4n-6, 22:6n-3 and 24:5n-3.The FA substrates were added to the yeast cultures at final concentrations of 0.5 (C 18 ), 0.75 (C 20 ), 1.0 (C 22 ) and 1.2 (C 24 ) mM to compensate for decreased efficiency of uptake with increased chain length [47].
To test the ability of the octopus Elovl4 to elongate saturated FAs, yeast transformed with pYES2-Elovl4 or pYES2 (negative control) were grown in the absence of exogenously added substrates and the capability of the Elovl4 to elongate saturated FAs was estimated by comparing the saturated FA profiles of both transformants.Three different replicates for each treatment were established.

Statistical Analyses
For the functional characterisation experiments (complementation and overexpression) of the octopus Scd, FA analyses from yeast samples were expressed as mean values ± standard deviation (n = 4).Similarly, the assay aiming to determine the ability of the octopus Elovl4 for elongation saturated FA was run in replicates (n = 3) and FA contents expressed as mean values ± standard deviation.Homogeneity of variances was checked by Barlett's test.Comparison of FA profiles from control and yeast expressing the O. vulgaris Scd (complementation and overexpression experiments) or Elovl4 were compared with a Student's t-test.Comparisons of the means with p values less or equal than 0.05 were considered significantly different.All the statistical analyses were carried out using the SPSS statistical package (SPSS Inc., Chicago, IL, USA).

Figure 1 .
Figure 1.Model of biosynthetic pathways of polyunsaturated fatty acids in cephalopods.Enzymatic activities shown in the diagram are predicted from heterologous expression in yeast (Saccharomyces cerevisiae) of fatty acyl desaturases (red arrows) and elongation of very long-chain fatty acid (Elovl) proteins (green arrows) from Octopus vulgaris [6,10] and Sepia officinalis [20].Dotted arrows indicate reactions that have not yet been demonstrated prior to the present study.β-ox, partial β-oxidation.

Figure 1 .
Figure 1.Model of biosynthetic pathways of polyunsaturated fatty acids in cephalopods.Enzymatic activities shown in the diagram are predicted from heterologous expression in yeast (Saccharomyces cerevisiae) of fatty acyl desaturases (red arrows) and elongation of very long-chain fatty acid (Elovl) proteins (green arrows) from Octopus vulgaris [6,10] and Sepia officinalis [20].Dotted arrows indicate reactions that have not yet been demonstrated prior to the present study.-ox,partial -oxidation.

Figure 2 .
Figure 2. ClustalW amino acid alignment comparing the Octopus vulgaris stearoyl-CoA desaturase (Scd) with homologous sequences from the Octopus bimaculoides (XP_014788514.1), the Caenorabditis elegans FAT-5 (NP_507482.1) and the C. elegans FAT-6 (NP_001255595.1), and a portion of the Saccharomyces cerevisiae Ole1p sequence.Identical residues are shaded black and similar residues (using ClustalW2 default parameters) are shaded grey.The three histidine boxes (HXXXH, HXXHH and QXXHH) are highlighted with grey squares.Four (I-IV) trans-membrane domains predicted by Watts and Browse [25] are underlined with dashed lines.

Figure 2 .
Figure 2. ClustalW amino acid alignment comparing the Octopus vulgaris stearoyl-CoA desaturase (Scd) with homologous sequences from the Octopus bimaculoides (XP_014788514.1), the Caenorabditis elegans FAT-5 (NP_507482.1) and the C. elegans FAT-6 (NP_001255595.1), and a portion of the Saccharomyces cerevisiae Ole1p sequence.Identical residues are shaded black and similar residues (using ClustalW2 default parameters) are shaded grey.The three histidine boxes (HXXXH, HXXHH and QXXHH) are highlighted with grey squares.Four (I-IV) trans-membrane domains predicted by Watts and Browse [25] are underlined with dashed lines.

Table 1 .
Fatty acid (FA) composition of the S. cerevisiae strain L8-14C transformed with the yeast-endogenous OLE1 (SC OLE1) or stearoyl-CoA desaturase from O. vulgaris (OV Scd).Results are expressed as an area percentage of total fatty acids (FAs) found in transformed yeast.Different letters for each FA or ratio indicate significant differences among treatments (t-test, p  0.05).

Table 2 .
Fatty acid (FA) composition of the S. cerevisiae InvSc1 transformed with either empty pYES2 vector (Control) or the common octopus Scd open reading frame (ORF).Different letters for each FA indicate significant differences among treatments (t-test, p  0.05).

Table 3 .
Role of the Octopus vulgaris Elovl4 in the biosynthesis of very long-chain (>C 24 ) polyunsaturated fatty acids (FAs).Conversions were calculated for each stepwise elongation according to the formula (areas of first product and longer chain products/(areas of all products with longer chain than substrate + substrate area)) ⇥ 100.The substrate FA varies as indicated in each step-wise elongation.