Greta Carmona-Antoñanzas

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.

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

BackgroundThe 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.ResultsPhylogenetic 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.ConclusionsWe 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.

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.

Molecular mechanism of dietary phospholipid requirement of Atlantic salmon, Salmo salar, fry.

The phospholipid (PL) requirement in fish is revealed by enhanced performance when larvae are provided PL-enriched diets. To elucidate the molecular mechanism underlying PL requirement in Atlantic salmon, Salmo salar, were fed a minimal PL diet and tissue samples from major lipid metabolic sites were dissected from fry and parr. In silico analysis and cloning techniques demonstrated that salmon possess a full set of enzymes for the endogenous production of PL. The gene expression data indicated that major PL biosynthetic genes of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylinositol (PtdIns) display lower expression in intestine during the early developmental stage (fry). This is consistent with the hypothesis that the intestine of salmon is immature at the early developmental stage with limited capacity for endogenous PL biosynthesis. The results also indicate that intact PtdCho, PtdEtn and PtdIns are required in the diet at this stage. PtdCho and sphingomyelin constitute the predominant PL in chylomicrons, involved in the transport of dietary lipids from the intestine to the rest of the body. As sphingomyelin can be produced from PtdCho in intestine of fry, our findings suggest that supplementation of dietary PtdCho alone during early developmental stages of Atlantic salmon would be sufficient to promote chylomicron formation. This would support efficient transport of dietary lipids, including PL precursors, from the intestine to the liver where biosynthesis of PtdEtn, PtdSer, and PtdIns is not compromised as in intestine facilitating efficient utilisation of dietary energy and the endogenous production of membrane PL for the rapidly growing and developing animal.

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.

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

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

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.

Light- and clock-control of genes involved in detoxification

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.

Ethanol toxicity differs depending on the time of day

ABSTRACT Circadian regulation of hepatic detoxification seems to be amongst the key roles of the biological clock. The liver is the major site for biotransformation, and in mammals, it contains several clock-controlled transcription factors such as proline and acidic amino acid-rich basic leucine zipper proteins (PAR bZIP) and basic-helix-loop-helix Per–Arnt–Sim (bHLH–PAS) family that act as circadian regulators of detoxification genes. This investigation explored the existence of daily and circadian expression of transcription factors involved in detoxification, as well as the temporal profile of a set of their target genes in zebrafish liver. In our study, zebrafish were able to synchronize to a light-dark (LD) cycle and displayed a diurnal pattern of activity. In addition, the expression of clock genes presented daily and circadian rhythmicity in liver. Apart from hlfa, the expression of PAR bZIP transcription factors also displayed daily rhythms, which appeared to be both light-dependent and clock-controlled, as circadian rhythms free-ran under constant conditions (continuous darkness, DD). Under LD, tefb, dbpa and dbpb expression peaked at the end of the darkness period whereas tefa showed peak levels of expression at the onset of the photophase. In addition, these four genes exhibited circadian expression under DD, with higher expression levels at the end of the subjective night. The expression of the bHLH-PAS transcription factor arh2 also showed circadian rhythmicity in zebrafish liver, peaking in the middle of the subjective night and approximately 3–4 h before peak expression of the PAR bZIP genes. Regarding the detoxification genes, the major target gene of AhR, cyp1a, showed daily and circadian expression with an acrophase 2 h after ahr2. Under LD, abcb4 also showed daily rhythmicity, with an acrophase 1–2 h after that of PAR bZIP factors during the transition between darkness and light phases, when zebrafish become active. However, the expression of six detoxification genes showed circadian rhythmicity under DD, including cyp1a and abcb4 as well as gstr1, mgst3a, abcg2 and sult2_st2. In all cases, the acrophases of these genes were found during the second half of the subjective night, in phase with the PAR bZIP transcription factors. This suggested that their expression is clock-controlled, either directly by core clock genes or through transcription factors. This study presents new data demonstrating that the process of detoxification is under circadian control in fish. Results showed that time of day should be considered when designing toxicological studies or administering drugs to fish.