H3K4me3 mediates uterine leiomyoma pathogenesis via neuronal processes, synapsis components, proliferation, and Wnt/ß-catenin and TGF-ß pathways.

BACKGROUND: Uterine leiomyomas (UL) are the most common benign tumor in women of reproductive age. Their pathology remains unclear, which hampers the development of safe and effective treatments. Raising evidence suggests epigenetics as a main mechanism involved in tumor development. Histone modification is a key component in the epigenetic regulation of gene expression. Specifically, the histone mark H3K4me3, which promotes gene expression, is altered in many tumors. In this study, we aimed to identify if the histone modification H3K4me3 regulates the expression of genes involved in uterine leiomyoma pathogenesis. METHODS: Prospective study integrating RNA-seq (n = 48) and H3K4me3 CHIP-seq (n = 19) data of uterine leiomyomas versus their adjacent myometrium. Differentially expressed genes (FDR < 0.01, log2FC > 1 or < - 1) were selected following DESeq2, edgeR, and limma analysis. Their differential methylation and functional enrichment (FDR < 0.05) were respectively analyzed with limma and ShinyGO. RESULTS: CHIP-seq data showed a global suppression of H3K4me3 in uterine leiomyomas versus their adjacent myometrial tissue (p-value< 2.2e-16). Integrating CHIP-seq and RNA-seq data highlighted that transcription of 696/922 uterine leiomyoma-related differentially expressed genes (DEG) (FDR < 0.01, log2FC > 1 or < - 1) was epigenetically mediated by H3K4me3. Further, 50 genes were differentially trimethylated (FDR < 0.05), including 33 hypertrimethylated/upregulated, and 17 hypotrimethylated/downregulated genes. Functional enrichment analysis of the latter showed dysregulation of neuron-related processes and synapsis-related cellular components in uterine leiomyomas, and a literature review study of these DEG found additional implications with tumorigenesis (i.e. aberrant proliferation, invasion, and dysregulation of Wnt/ß-catenin, and TGF-ß pathways). Finally, SATB2, DCX, SHOX2, ST8SIA2, CAPN6, and NPTX2 proto-oncogenes were identified among the hypertrimethylated/upregulated DEG, while KRT19, ABCA8, and HOXB4 tumor suppressor genes were identified among hypotrimethylated/downregulated DEG. CONCLUSIONS: H3K4me3 instabilities alter the expression of oncogenes and tumor suppressor genes, inducing aberrant proliferation, and dysregulated Wnt/ß-catenin, and TGF-ß pathways, that ultimately promote uterine leiomyoma progression. The reversal of these histone modifications may be a promising new therapeutic alternative for uterine leiomyoma patients.

Investigation of deltamethrin resistance in salmon lice (Lepeophtheirus salmonis) provides no evidence for roles of mutations in voltage-gated sodium channels.

BACKGROUND: The pyrethroid deltamethrin is used to treat infestations of farmed salmon by parasitic salmon lice, Lepeophtheirus salmonis (Krøyer). However, the efficacy of deltamethrin for salmon delousing is threatened by resistance development. In terrestrial arthropods, knockdown resistance (kdr) mutations of the voltage-gated sodium channel (Nav ), the molecular target for pyrethroids, can cause deltamethrin resistance. A putative kdr mutation of an L. salmonis sodium channel homologue (LsNav 1.3 I936V) has been identified previously. At the same time, deltamethrin resistance of L. salmonis has been shown to be inherited maternally and to be associated with mitochondrial DNA (mtDNA) mutations. This study assessed potential roles of the above putative kdr mutation as a determinant of deltamethrin resistance in laboratory strains and field populations of L. salmonis. RESULTS: The deltamethrin-resistant L. salmonis strain IoA-02 expresses the LsNav 1.3 I936V mutation but was susceptible to the non-ester pyrethroid etofenprox, a compound against which pyrethroid-resistant arthropods are usually cross-resistant if resistance is caused by Nav mutations. In a family derived from a cross between an IoA-02 male and a drug-susceptible female lacking the kdr mutation, deltamethrin resistance was not associated with the genotype at the LsNav 1.3 locus (P > 0.05). Similarly, in Scottish field populations of L. salmonis, LsNav 1.3 I936V showed no association with deltamethrin resistance. By contrast, genotypes at the mtDNA loci A14013G and A9030G were significantly associated with deltamethrin resistance (P < 0.001). CONCLUSION: In the studied L. salmonis isolates, deltamethrin resistance was unrelated to the LsNav 1.3 I936V mutation, but showed close association with mtDNA mutations.

Genome-wide survey of cytochrome P450 genes in the salmon louse Lepeophtheirus salmonis (Krøyer, 1837).

BACKGROUND: The salmon louse (Lepeophtheirus salmonis) infests farmed and wild salmonid fishes, causing considerable economic damage to the salmon farming industry. Infestations of farmed salmon are controlled using a combination of non-medicinal approaches and veterinary drug treatments. While L. salmonis has developed resistance to most available salmon delousing agents, relatively little is known about the molecular mechanisms involved. Members of the cytochrome P450 (CYP) superfamily are typically monooxygenases, some of which are involved in the biosynthesis and metabolism of endogenous compounds, while others have central roles in the detoxification of xenobiotics. In terrestrial arthropods, insecticide resistance can be based on the enhanced expression of CYPs. The reported research aimed to characterise the CYP superfamily in L. salmonis and assess its potential roles in drug resistance. METHODS: Lepeophtheirus salmonis CYPs were identified by homology searches of the genome and transcriptome of the parasite. CYP transcript abundance in drug susceptible and multi-resistant L. salmonis was assessed by quantitative reverse transcription PCR, taking into account both constitutive expression and expression in parasites exposed to sublethal levels of salmon delousing agents, ecdysteroids and environmental chemicals. RESULTS: The above strategy led to the identification of 25 CYP genes/pseudogenes in L. salmonis, making its CYP superfamily the most compact characterised for any arthropod to date. Lepeophtheirus salmonis possesses homologues of a number of arthropod CYP genes with roles in ecdysteroid metabolism, such as the fruit fly genes disembodied, shadow, shade, spook and Cyp18a1. CYP transcript expression did not differ between one drug susceptible and one multi-resistant strain of L. salmonis. Exposure of L. salmonis to emamectin benzoate or deltamethrin caused the transcriptional upregulation of certain CYPs. In contrast, neither ecdysteroid nor benzo[a]pyrene exposure affected CYP transcription significantly. CONCLUSIONS: The parasite L. salmonis is demonstrated to possess the most compact CYP superfamily characterised for any arthropod to date. The complement of CYP genes in L. salmonis includes conserved CYP genes involved in ecdysteroid biosynthesis and metabolism, as well as drug-inducible CYP genes. The present study does not provide evidence for a role of CYP genes in the decreased susceptibility of the multiresistant parasite strain studied.

Correction: Earlier relapse detection after allogeneic haematopoietic stem cell transplantation by chimerism assays: Digital PCR versus quantitative real-time PCR of insertion/deletion polymorphisms.

[This corrects the article DOI: 10.1371/journal.pone.0212708.].

Earlier relapse detection after allogeneic haematopoietic stem cell transplantation by chimerism assays: Digital PCR versus quantitative real-time PCR of insertion/deletion polymorphisms.

BACKGROUND: The analysis of molecular haematopoietic chimerisms (HC) has become a well-established method to monitor the transplant evolution and to assess the risk of relapse after allogeneic stem cells transplantation (allo-STC). Different techniques and molecular markers are being used for chimerism surveillance after transplantation, including quantitative real-time PCR (qPCR) and the recently developed digital PCR (dPCR). This study aims to compare the sensitivity and accuracy of both methods to quantify HC and predict early relapse. METHODOLOGY: HC was evaluated using custom PCR systems for the specific detection of the Y-chromosome, null alleles and insertion-deletion polymorphisms. A total of 281 samples from 28 adult patients who underwent an allo-SCT were studied. Increasing mixed chimerism was detected prior to relapse in 100% of patients (18 relapses). RESULTS: Compared with conventional qPCR amplification, dPCR predicted relapse with a median anticipation period of 63 days versus 45.5 days by qPCR. Overall, 56% of the relapses were predicted earlier with dPCR whereas 38% of the relapses where detected simultaneously using both techniques and only in 1 case, relapse was predicted earlier with qPCR. CONCLUSIONS: In conclusion, chimerism determination by dPCR constitutes a suitable technique for the follow-up of patients with haematological pathologies after allo-STC, showing greater sensitivity to predict an early relapse.

Mutations in voltage-gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis).

BACKGROUND: Parasitic salmon lice (Lepeophtheirus salmonis) cause high economic losses in Atlantic salmon farming. Pyrethroids, which block arthropod voltage-gated sodium channels (Nav 1), are used for salmon delousing. However, pyrethroid resistance is common in L. salmonis. The present study characterized Nav 1 homologues in L. salmonis in order to identify channel mutations associated to resistance, called kdr (knockdown) mutations. RESULTS: Genome scans identified three L. salmonis Nav 1 homologues, LsNav 1.1, LsNav 1.2 and LsNav 1.3. Arthropod kdr mutations map to specific Nav 1 regions within domains DI-III, namely segments S5 and S6 and the linker helix connecting S4 and S5. The above channel regions were amplified by RT-PCR and sequenced in deltamethrin-susceptible and deltamethrin-resistant L. salmonis. While LsNav 1.1 and LsNav 1.2 lacked nucleotide polymorphisms showing association to resistance, LsNav 1.3 showed a non-synonymous mutation in S5 of DII occurring in deltamethrin-resistant parasites. The mutation is homologous to a previously described kdr mutation (I936V, numbering according to Musca domestica Vssc1) and was present in two pyrethroid-resistant L. salmonis strains (allele frequencies of 0.800 and 0.357), but absent in two pyrethroid-susceptible strains. CONCLUSIONS: The present study indicates that a kdr-mutation in LsNaV 1.3 may contribute to deltamethrin resistance in L. salmonis. © 2018 Society of Chemical Industry.

Ethanol toxicity differs depending on the time of day.

Ethanol is one of the most commonly abused drugs and consequently its toxic and psychoactive effect has been widely investigated, although little is known about the time-dependent effects of this drug. In the present research zebrafish was used to assess daily rhythms in ethanol toxicity and behavioural effects, as well as the temporal pattern of expression of key genes involved in ethanol detoxification in the liver (adh8a, adh5, aldh2.1 and aldh2.2). Our results showed marked differences in the mortality rate of zebrafish larvae depending on the time of day of the exposure to 5% ethanol for 1h (82% and 6% mortality in the morning and at night, respectively). A significant daily rhythm was detected with the acrophase located at "zeitgeber" time (ZT) = 04:22 h. Behavioural tests exposing zebrafish to 1% ethanol provoked a major decrease in swimming activity (68-84.2% reduction) at ZT2, ZT6 and ZT10. In contrast, exposure at ZT18 stimulated swimming activity (27% increase). During the day fish moved towards the bottom of the tank during ethanol exposure, whereas at night zebrafish increased their activity levels right after the exposure to ethanol. Genes involved in ethanol detoxification failed to show significant daily rhythms in LD, although all of them exhibited circadian regulation in constant darkness (DD) with acrophases in phase and located at the end of the subjective night. Taken altogether, this research revealed the importance of considering the time of day when designing and carrying out toxicological and behavioural tests to investigate the effects of ethanol, as the adverse effects of this drug were more marked when fish were exposed in the morning than at night.

Maternal inheritance of deltamethrin resistance in the salmon louse Lepeophtheirus salmonis (Krøyer) is associated with unique mtDNA haplotypes.

Parasitic infections by the salmon louse, Lepeophtheirus salmonis (Krøyer), cause huge economic damage in salmon farming in the northern hemisphere, with combined treatment costs and production losses in 2014 having been estimated at US$ 350 million for Norway (annual production 1.25 million tonnes). The control of L. salmonis relies significantly on medicinal treatments, supplemented by non-pharmacological approaches. However, efficacy losses have been reported for several delousing agents, including the pyrethroid deltamethrin. The aim of the present study was to analyse the genetic basis of deltamethrin resistance in L. salmonis. Deltamethrin median effective concentrations (EC50) were 0.28 µg L-1 in the drug susceptible L. salmonis strain IoA-00 and 40.1 µg L-1 in the pyrethroid resistant strain IoA-02. IoA-00 and IoA-02 were crossed to produce families spanning one parental and three filial generations (P0, F1-F3). In three families derived from P0 crosses between an IoA-00 sire and an IoA-02 dam, 98.8% of F2 parasites (n = 173) were resistant, i.e. remained unaffected after exposure to 2.0 µg L-1 deltamethrin. F3 parasites from these crosses showed a deltamethrin EC50 of 9.66 µg L-1. In two families of the inverse orientation at P0 (IoA-02 sire x IoA-00 dam), 16.7% of F2 parasites were resistant (n = 84), while the deltamethrin EC50 in F3 animals was 0.26 µg L-1. The results revealed a predominantly maternal inheritance of deltamethrin resistance. The 15,947-nt mitochondrial genome was sequenced and compared among six unrelated L. salmonis strains and parasites sampled from wild salmon in 2010. IoA-02 and three further deltamethrin resistant strains, established from isolates originating from different regions of Scotland, showed almost identical mitochondrial haplotypes. In contrast, the mitochondrial genome was variable among susceptible strains and L. salmonis from wild hosts. Deltamethrin caused toxicity and depletion of whole body ATP levels in IoA-00 but not IoA-02 parasites. The maternal inheritance of deltamethrin resistance and its association with mitochondrial haplotypes suggests that pyrethroid toxicity in L. salmonis may involve molecular targets encoded by mitochondrial genes.

Two alternative pathways for docosahexaenoic acid (DHA, 22:6n-3) biosynthesis are widespread among teleost fish.

Docosahexaenoic acid (DHA) plays important physiological roles in vertebrates. Studies in rats and rainbow trout confirmed that DHA biosynthesis proceeds through the so-called "Sprecher pathway", a biosynthetic process requiring a Δ6 desaturation of 24:5n-3 to 24:6n-3. Alternatively, some teleosts possess fatty acyl desaturases 2 (Fads2) that enable them to biosynthesis DHA through a more direct route termed the "Δ4 pathway". In order to elucidate the prevalence of both pathways among teleosts, we investigated the Δ6 ability towards C24 substrates of Fads2 from fish with different evolutionary and ecological backgrounds. Subsequently, we retrieved public databases to identify Fads2 containing the YXXN domain responsible for the Δ4 desaturase function, and consequently enabling these species to operate the Δ4 pathway. We demonstrated that, with the exception of Δ4 desaturases, fish Fads2 have the ability to operate as Δ6 desaturases towards C24 PUFA enabling them to synthesise DHA through the Sprecher pathway. Nevertheless, the Δ4 pathway represents an alternative route in some teleosts and we identified the presence of putative Δ4 Fads2 in a further 11 species and confirmed the function as Δ4 desaturases of Fads2 from medaka and Nile tilapia. Our results demonstrated that two alternative pathways for DHA biosynthesis exist in teleosts.

Time-to-response toxicity analysis as a method for drug susceptibility assessment in salmon lice.

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite causing infections of wild and farmed Atlantic salmon (Salmo salar L.) in the Northern hemisphere. While L. salmonis control at commercial mariculture sites increasingly employs non-medicinal approaches, such as cage designs reducing infection rates and biological control through cleaner fish, anti-parasitic drugs are still a requirement for effective fish health care. With only a limited range of salmon delousing agents available, all of which have been in use for more than a decade, drug resistance formation has been reported for different products. Successful resistance management requires reliable susceptibility assessment, which is usually achieved through L. salmonis bioassays. These tests involve the exposure of parasites to different drug concentrations and require significant numbers of suitable L. salmonis stages. The present study reports an alternative bioassay that is based on time-to-response toxicity analyses and can be carried out with limited parasite numbers. The assay determines the median effective time (ET50), i.e., the time required until impaired swimming and/or attachment behaviour becomes apparent in 50% of parasites, by conducting repeated examinations of test animals starting at the time point where exposure to a set drug concentration commences. This experimental approach further allows the estimation of the apparent drug susceptibility of individual L. salmonis by determining their time to response, which may prove useful in experiments designed to elucidate associations between genetic factors and the drug susceptibility phenotype of parasites. Three laboratory strains of L. salmonis differing in susceptibility to emamectin benzoate were characterised using standard 24 h bioassays and time-to-response toxicity assays. While both the median effective concentration (EC50) and the ET50 showed variability between experimental repeats, both types of bioassay consistently discriminated susceptible and drug-resistant L. salmonis laboratory strains. STATEMENT OF RELEVANCE: Infections by sea lice cause significant costs to the global salmon farming industry, which have been estimated to exceed €300 million per year worldwide. Control of sea lice still relies to a significant extent on chemical delousing; however, chemical control is threatened by resistance formation. Resistance can be combated by rotation between different drugs and strategic implementation of non-medicinal strategies. However, resistance management requires reliable and feasible methods of susceptibility assessment. The present study is a technical note introducing a novel approach to susceptibility assessments in sea lice. The method can be applied in susceptibility assessments on farms, where it offers the advantage of a reduced requirement of parasites for testing. In addition, the novel method allows deriving the times of parasite require to show a response after drug treatment has started, thus providing a variable characterizing the drug susceptibility phenotype of individual parasites. Accordingly, the bioassay approach presented here will be useful for studies aiming at unravelling the genetic determinants of drug resistance.

Regulatory divergence of homeologous Atlantic salmon elovl5 genes following the salmonid-specific whole-genome duplication.

Fatty acyl elongase 5 (elovl5) is a critical enzyme in the vertebrate biosynthetic pathway which produces the physiologically essential long-chain polyunsaturated fatty acids (LC-PUFA), docosahexenoic acid (DHA), and eicosapentenoic acid (EPA) from 18 carbon fatty acids precursors. In contrast to most other vertebrates, Atlantic salmon possess two copies of elovl5 (elovl5a and elovl5b) as a result of a whole-genome duplication (WGD) which occurred at the base of the salmonid lineage. WGDs have had a major influence on vertebrate evolution, providing extra genetic material, enabling neofunctionalization to accelerate adaptation and speciation. However, little is known about the mechanisms by which such duplicated homeologous genes diverge. Here we show that homeologous Atlantic salmon elovl5a and elovl5b genes have been asymmetrically colonised by transposon-like elements. Identical locations and identities of insertions are also present in the rainbow trout duplicate elovl5 genes, but not in the nearest extant representative preduplicated teleost, the northern pike. Both elovl5 salmon duplicates possessed conserved regulatory elements that promoted Srebp1- and Srebp2-dependent transcription, and differences in the magnitude of Srebp response between promoters could be attributed to a tandem duplication of SRE and NF-Y cofactor binding sites in elovl5b. Furthermore, an insertion in the promoter region of elovl5a confers responsiveness to Lxr/Rxr transcriptional activation. Our results indicate that most, but not all, transposon mobilisation into elovl5 genes occurred after the split from the common ancestor of pike and salmon, but before more recent salmonid speciations, and that divergence of elovl5 regulatory regions have enabled neofuntionalization by promoting differential expression of these homeologous genes.

Isolation and Functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 Desaturase Activity.

Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C18 precursors.

A Survey of the ATP-Binding Cassette (ABC) Gene Superfamily in the Salmon Louse (Lepeophtheirus salmonis).

Salmon lice, Lepeophtheirus salmonis (Krøyer, 1837), are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon, Salmo salar Linnaeus, 1758. The control of L. salmonis at fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which in L. salmonis is documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC) gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters in L. salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes in L. salmonis for which, ABC superfamily members were identified through homology searching of the L. salmonis genome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq) of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters, i.e. subfamilies B (4 sequences), C (11) and G (2). The results suggest that the ABC gene family of L. salmonis possesses fewer members than recorded for other arthropods. The present survey of the L. salmonis ABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance.

Conservation of lipid metabolic gene transcriptional regulatory networks in fish and mammals.

Lipid content and composition in aquafeeds have changed rapidly as a result of the recent drive to replace ecologically limited marine ingredients, fishmeal and fish oil (FO). Terrestrial plant products are the most economic and sustainable alternative; however, plant meals and oils are devoid of physiologically important cholesterol and long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic (EPA), docosahexaenoic (DHA) and arachidonic (ARA) acids. Although replacement of dietary FO with vegetable oil (VO) has little effect on growth in Atlantic salmon (Salmo salar), several studies have shown major effects on the activity and expression of genes involved in lipid homeostasis. In vertebrates, sterols and LC-PUFA play crucial roles in lipid metabolism by direct interaction with lipid-sensing transcription factors (TFs) and consequent regulation of target genes. The primary aim of the present study was to elucidate the role of key TFs in the transcriptional regulation of lipid metabolism in fish by transfection and overexpression of TFs. The results show that the expression of genes of LC-PUFA biosynthesis (elovl and fads2) and cholesterol metabolism (abca1) are regulated by Lxr and Srebp TFs in salmon, indicating highly conserved regulatory mechanism across vertebrates. In addition, srebp1 and srebp2 mRNA respond to replacement of dietary FO with VO. Thus, Atlantic salmon adjust lipid metabolism in response to dietary lipid composition through the transcriptional regulation of gene expression. It may be possible to further increase efficient and effective use of sustainable alternatives to marine products in aquaculture by considering these important molecular interactions when formulating diets.

An evolutionary perspective on Elovl5 fatty acid elongase: comparison of Northern pike and duplicated paralogs from Atlantic salmon.

BACKGROUND: The ability to produce physiologically critical LC-PUFA from dietary fatty acids differs greatly among teleost species, and is dependent on the possession and expression of fatty acyl desaturase and elongase genes. Atlantic salmon, as a result of a recently duplicated genome, have more of these enzymes than other fish. Recent phylogenetic studies show that Northern pike represents the closest extant relative of the preduplicated ancestral salmonid. Here we characterise a pike fatty acyl elongase, elovl5, and compare it to Atlantic salmon elovl5a and elovl5b duplicates. RESULTS: Phylogenetic analyses show that Atlantic salmon paralogs are evolving symmetrically, and they have been retained in the genome by purifying selection. Heterologous expression in yeast showed that Northern pike Elovl5 activity is indistinguishable from that of the salmon paralogs, efficiently elongating C18 and C20 substrates. However, in contrast to salmon, pike elovl5 was predominantly expressed in brain with negligible expression in liver and intestine. CONCLUSIONS: We suggest that the predominant expression of Elovl5b in salmon liver and Elovl5a in salmon intestine is an adaptation, enabled by genome duplication, to a diet rich in terrestrial invertebrates which are relatively poor in LC-PUFA. Pike have retained an ancestral expression profile which supports the maintenance of PUFA in the brain but, due to a highly piscivorous LC-PUFA-rich diet, is not required in liver and intestine. Thus, the characterisation of elovl5 in Northern pike provides insights into the evolutionary divergence of duplicated genes, and the ecological adaptations of salmonids which have enabled colonisation of nutrient poor freshwaters.

Biosynthesis of very long-chain fatty acids (C>24) in Atlantic salmon: cloning, functional characterisation, and tissue distribution of an Elovl4 elongase.

The elongases of very long-chain fatty acids (Elovl) account for the rate-limiting condensation step of the elongation process in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovl family, Elovl4, has been regarded as a critical enzyme in vertebrates in the production of the so-called very long-chain fatty acids (VLC-FA), a group of compounds that has been scarcely explored in fish. Here we report on the cloning of a novel Elovl4-like elongase from Atlantic salmon (Salmo salar). The salmon Elovl4 cDNA codes for a putative protein containing 306 amino acids. Heterologous expression in yeast demonstrated that salmon Elovl4 efficiently elongated saturated FAs up to 36:0, with 24:0 and 26:0 appearing as preferred substrates. Additionally, salmon Elovl4 effectively converted C20 and C22 polyunsaturated fatty acids to elongated polyenoic products up to C36. Tissue distribution showed that Elovl4 mRNA transcripts are abundant in eye, brain and testes, suggesting that, as described in mammals, these tissues are important metabolic sites for the biosynthesis of VLC-FA. Our results are discussed in comparison with the functional analyses observed in Elovl4 proteins from other vertebrates, and also other Elovl proteins investigated previously in Atlantic salmon.