Diseño y optimización de liposomas para un uso como sistema de suministro de nutrientes a larvas de peces marinos.

RESUMEN Las deficiencias nutricionales de las presas vivas usadas como primer alimento exógeno en el cultivo de larvas de peces marinos hacen necesaria la suplementación de las dietas con nutrientes que satisfagan los requerimientos de las larvas. Los liposomas constituyen una herramienta con gran potencial ya que pueden formularse con nutrientes hidrosolubles disueltos en su fase acuosa y liposolubles inmersos en el ambiente lipofílico entre las cadenas de ácidos grasos de los fosfolípidos. Este estudio valora el empleo de liposomas para suministrar nutrientes a larvas de peces marinos a través de dos vías. La primera plantea la vehiculación de nutrientes a larvas de peces a través de la bioencapsulación de liposomas ricos en nutrientes esenciales en nauplios de Artemia. La segunda alternativa está relacionada con la administración directa a las larvas mediante inmersiones de éstas en suspensiones de liposomas ricos en nutrientes. Liposomas como enriquecedores de Artemia El estudio se inicia con un análisis preliminar sobre la integridad de los liposomas en condiciones de enriquecimiento de Artemia. Los resultados indican que los liposomas liberan parte de las sustancias disueltas en su fase acuosa interna. En cuanto a la estabilidad química, los resultados muestran que los liposomas son menos susceptibles a la peroxidación que otros enriquecedores convencionales. Por último, el estudio sobre el cambio de tamaño de los liposomas durante el enriquecimiento indica que no muestran alteraciones importantes que puedan comprometer su rendimiento como producto enriquecedor. A la vista de estos resultados preliminares, se escogieron unas formulaciones de liposomas para su utilización en el enriquecimiento de Artemia en ácidos grasos esenciales (EFAs), vitaminas (A y C) y aminoácidos. La mejora del contenido en EFAs de nauplios de Artemia se llevó a cabo con liposomas formulados con fosfolípidos marinos. Los resultados muestran que estos liposomas pueden igualar los contenidos de EFAs de los nauplios enriquecidos con productos comerciales de uso habitual. Además, se presenta un protocolo óptimo de utilización para maximizar los niveles de EFAs en los nauplios. Como ya se ha comentado anteriormente, los liposomas poseen una estructura que permite la inclusión, junto a los fosfolípidos, de otros nutrientes esenciales como las vitaminas y los aminoácidos. Los resultados revelan la capacidad de liposomas para aumentar el contenido en vitamina A de los nauplios. En cuanto a la vehiculación de nutrientes hidrosolubles, los experimentos realizados muestran que los liposomas unilamelares tienen una escasa eficacia enriquecedora en este tipo de sustancias. No obstante, los liposomas de tipo multilamelar exhibieron una mayor eficacia logrando aumentar los niveles de metionina (hidrosoluble) libre de los nauplios en comparación a nauplios tratados con metionina simplemente disuelta. El estudio sobre la utilización de liposomas como enriquecedores de Artemia se completa con la realización de experimentos de alimentación larvaria. Estos ensayos demuestran la posibilidad de utilizar nauplios enriquecidos con liposomas como alimento de larvas de peces marinos sin que se observen diferencias en la supervivencia y el crecimiento obtenidos con respecto a tratamientos con un producto comercial de eficacia contrastada. Suministro directo de nutrientes a larvas mediante liposomas Se realizaron inmersiones de larvas de peces con liposomas ricos en nutrientes esenciales. Los resultados evidencian la posibilidad de tratar larvas de peces marinos con liposomas formulados con nutrientes esenciales de naturaleza diversa sin detrimento de la supervivencia de los animales. Sin embargo, la utilidad de esta vía de administración de nutrientes no ha sido demostrada a pesar de los numerosos mecanismos de interacción larva-liposoma implicados. La utilización de nutrientes marcados, así como la determinación de índices de condición nutricional que constaten las ventajas nutricionales derivadas de la incorporación de los nutrientes liposomados, se vislumbran como líneas futuras de investigación. __________________________________________________________________________________________________Nutritional deficiencies of live preys used as first exogenous feeding in the rearing of marine fish larvae make necessary the supplementation of the diets with certain nutrients in order to fulfill the requirements of the fish. In most cases, larval nutrition studies have been focused on the essential fatty acid demands, but other compounds as phospholipids, vitamins and free amino acids have been proposed as crucial elements in the early development stages of several species of marine fish. The present study assesses the use of liposomes, phospholipid vesicles enclosing an internal aqueous space, for the delivery of both lipophilic and hydrophilic nutrients to marine fish larvae. Two different administration ways were studied. Firstly, liposomes were used as an enrichment product of live preys. Secondly, a direct system was assessed by immersing marine fish larvae in liposome suspensions. Bioencapsulation of liposomes in Artemia nauplii Several liposome formulations were employed for the enrichment of Artemia nauplii in some essential nutrients such as fatty acids, vitamins and amino acids. Liposomes formulated with marine phospholipids demonstrated the improvement of the essential fatty acid content in the nauplii, and an optimal protocol of use with this product was established. Additionally, our results showed that liposomes are useful tools for the enrichment of Artemia in both liposoluble (vitamin A) and hydrosoluble compounds (methionine). Immersion of marine fish larvae in liposome suspensions A novel technique to transfer nutrients to fish larvae consists of the immersion of larvae in liposomated substances, obviating their bioencapsulation in the live preys and avoiding their consequent metabolic activity over the administered substances. Several studies were carried out with the purpose of detecting the incorporation of lipophilic (EFA) and hydrophilic (ascorbate and methionine) nutrients into marine larvae by means of liposome baths. Results would indicate a low entry of liposomated nutrients into the larvae, although analyses seem to reflect slightly higher average contents of EFA and ascorbate nutrient in larvae treated with liposomes compared to control non-treated larvae. These results could be confirmed optimizing the experimental designs

Dietary Effect on the Proteome of the Common Octopus (Octopus vulgaris) Paralarvae

Nowadays, the common octopus (Octopus vulgaris) culture is hampered by massive mortalities occurring during early life-cycle stages (paralarvae). Despite the causes of the high paralarvae mortality are not yet well-defined and understood, the nutritional stress caused by inadequate diets is pointed out as one of the main factors. In this study, the effects of diet on paralarvae is analyzed through a proteomic approach, to search for novel biomarkers of nutritional stress. A total of 43 proteins showing differential expression in the different conditions studied have been identified. The analysis highlights proteins related with the carbohydrate metabolism: glyceraldehyde-3-phosphate-dedydrogenase (GAPDH), triosephosphate isomerase; other ways of energetic metabolism: NADP+-specific isocitrate dehydrogenase, arginine kinase; detoxification: glutathione-S-transferase (GST); stress: heat shock proteins (HSP70); structural constituent of eye lens: S-crystallin 3; and cytoskeleton: actin, actin-beta/gamma1, beta actin. These results allow defining characteristic proteomes of paralarvae depending on the diet; as well as the use of several of these proteins as novel biomarkers to evaluate their welfare linked to nutritional stress. Notably, the changes of proteins like S-crystallin 3, arginine kinase and NAD+ specific isocitrate dehydrogenase, may be related to fed vs. starving paralarvae, particularly in the first 4 days of development.En prens

Studies on the characterisation of biomarkers of nutritionally-derived stress in paralarval cultures of the common octopus (Octopus vulgaris)

Comunicación presentada en las CephsInAction MC & WGs meetings, celebradas en Barcelona, España, del 14 al 16 de marzo de 2014 Nowadays, due to the high mortality within the first 30 days of life, octopus paralarvae culture represents the main obstacle for commercial production of this species. The causes of such mortality are not yet well defined and understood. As a part of a broader project aimed at characterising the causes of such massive mortality, we envisaged the study of nutritionally-derived stress, through the selection of biomarkers capable of its detection and quantification.This research was supported by “Ministerio de Ciencia e Innovación” through the OCTOPHYS Project (AGL-2010-22120-C03-02), and by a Marie Curie R.G. (PERG08-GA-2010-276916, LONGFA). The study was also carried out under the scope of a PROMETEO Project (2010/006) from Generalitat Valenciana.Peer Reviewe

Antioxidant activity and lipid peroxidation in Artemia nauplii enriched with a DHA-rich oil emulsion and the effect of adding an external antioxidant based on hydroxytyrosol

Artemia nauplii catabolise polyunsaturated fatty acids (PUFA); in particular, they retroconvert docosahexaenoic acid (DHA, 22:6n-3), so enrichment is a continuous quest towards increasing PUFA through the use of PUFA-rich enrichment products. However, optimal conditions during enrichment (aeration, illumination and temperatures around 28 ºC) tend to accelerate autoxidation of PUFA, and the formation of potentially toxic oxidation products. Water-soluble antioxidants like the polyphenolic compound hydroxytyrosol (3,4-dihydroxyphenylethanol), a polar molecule found in the water fraction resulting after the milling process of olives, arise as promising compounds to prevent oxidation during Artemia enrichments. We investigated the antioxidant activity and lipid peroxidation in Artemia nauplii during enrichment, and the effect of adding an external antioxidant based on hydroxytyrosol during the enrichment with a PUFA-rich emulsion (M70). For this purpose, the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase), as well as lipid peroxidation, were determined in enriched and unenriched Artemia nauplii. To validate antioxidant activity and lipid peroxidation, in a first experiment, nauplii were enriched with microalgae (Tetraselmis suecica), yeast (Saccharomyces cerevisiae) and M70 emulsion. In a second experiment, enrichment with a commercial emulsion (DC Super Selco), M70, and a combination of M70 and hydroxytyrosol (Hytolive, HYT) added as an external antioxidant was performed. The combination of M70 with HYT produced the best results, in terms of activity of antioxidant enzymes. The analysis of the fatty acids from total lipids showed that the addition of hydroxytyrosol preserved the DHA percentage of enriched nauplii

Pparγ Is Involved in the Transcriptional Regulation of Liver LC-PUFA Biosynthesis by Targeting the Δ6Δ5 Fatty Acyl Desaturase Gene in the Marine Teleost Siganus canaliculatus

As the first marine teleost demonstrated to have the ability of long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis from C18 PUFA precursors, the rabbitfish Siganus canaliculatus provides us a unique model for clarifying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts aiming at the replacement of dietary fish oil (rich in LC-PUFA) with vegetable oils (rich in C18 PUFA precursors but devoid of LC-PUFA). In the study of transcription regulation of gene encoding the Δ6Δ5 fatty acyl desaturase (Δ6Δ5 Fads), a rate-limiting enzyme catalyzing the first step of LC-PUFA biosynthesis in rabbitfish, a binding site for the transcription factor (TF), peroxisome proliferator-activated receptor γ (Pparγ), was predicted in Δ6Δ5 fads2 promoter by bioinformatics analysis, and thus the present study focused on the regulatory roles of Pparγ on Δ6Δ5 fads2. First, the activity of the Δ6Δ5 fads2 promoter was proved to be downregulated by pparγ overexpression and upregulated by treatment of Pparγ antagonist (GW9662), respectively, in HEK 293T cells with the dual luciferase reporter assay. Pparγ was further confirmed to interact with the promoter by electrophoretic mobility shift assay. Moreover, in S. canaliculatus hepatocyte line (SCHL) cells, GW9662 decreased the expression of pparγ together with increase of Δ6Δ5 fads2 mRNA. Besides, Δ6Δ5 fads2 expression was increased by pparγ RNAi knockdown and reduced by its mRNA overexpression. Furthermore, knockdown of pparγ induced a high conversion of 18:3n−3 to 18:4n−3 and 18:2n−6 to 18:3n−6, while pparγ mRNA overexpression led to a lower conversion of that, and finally a significant decrease of 20:4n-6(ARA), 20:5n-3(EPA), and 22:6n-3(DHA) production. The results indicate that Pparγ is involved in the transcriptional regulation of liver LC-PUFA biosynthesis by targeting Δ6Δ5 fads2 in rabbitfish, which is the first report of Pparγ involvement in the regulation of LC-PUFA biosynthesis in teleosts

Molecular and Functional Characterization of Elovl4 Genes in Sparus aurata and Solea senegalensis Pointing to a Critical Role in Very Long-Chain (>C24) Fatty Acid Synthesis during Early Neural Development of Fish

Very long-chain fatty acids (VLC-FA) play critical roles in neural tissues during the early development of vertebrates. However, studies on VLC-FA in fish are scarce. The biosynthesis of VLC-FA is mediated by elongation of very long-chain fatty acid 4 (Elovl4) proteins and, consequently, the complement and activity of these enzymes determines the capacity that a given species has for satisfying its physiological demands, in particular for the correct development of neurophysiological functions. The present study aimed to characterize and localize the expression of elovl4 genes from Sparus aurata and Solea senegalensis, as well as to determine the function of their encoded proteins. The results confirmed that both fish possess two distinct elovl4 genes, named elovl4a and elovl4b. Functional assays demonstrated that both Elovl4 isoforms had the capability to elongate long-chain (C20–24), both saturated (SFA) and polyunsaturated (PUFA), fatty acid precursors to VLC-FA. In spite of their overlapping activity, Elovl4a was more active in VLC-SFA elongation, while Elovl4b had a preponderant elongation activity towards n-3 PUFA substrates, particularly in S. aurata, being additionally the only isoform that is capable of elongating docosahexaenoic acid (DHA). A preferential expression of elovl4 genes was measured in neural tissues, being elovl4a and elovl4b mRNAs mostly found in brain and eyes, respectively.info:eu-repo/semantics/publishedVersio

Biosynthesis of polyunsaturated fatty acids in marine invertebrates: Recent advances in molecular mechanisms

Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs

Dietary DHA/EPA ratio affected tissue fatty acid profiles, antioxidant capacity, hematological characteristics and expression of lipid-related genes but not growth in juvenile black seabream (Acanthopagrus schlegelii)

An 8-week feeding trial was conducted to investigate the effects of dietary docosahexaenoic to eicosapentaenoic acid ratio (DHA/EPA) on growth performance, fatty acid profiles, anti- oxidant capacity, hematological characteristics and expression of some lipid metabolism related genes of juvenile black seabream (Acanthopagrus schlegelii) of initial weight 9.47 ± 0.03 g. Five isonitrogenous and isolipidic diets (45% crude protein and 14% crude lipid) were formulated to contain graded DHA/EPA ratios of 0.65, 1.16, 1.60, 2.03 and 2.67. There were no differences in growth performance and feed utilization among treatments. Fish fed higher DHA/EPA ratios had higher malondialdehyde (MDA) contents in serum than lower ratios. Serum triacylglycerol (TAG) content was significantly higher in fish fed the lowest DHA/EPA ratio. Tissue fatty acid profiles reflected the diets despite down-regulation of LC- PUFA biosynthesis genes, fatty acyl desaturase 2 (fads2) and elongase of very long-chain fatty acids 5 (elovl5), by high DHA/EPA ratios. Expression of acetyl-CoA carboxylase alpha (accα) and carnitine palmitoyl transferase 1A (cpt1a) were up-regulated by high DHA/EPA ratio, whereas sterol regulatory element-binding protein-1 (srebp-1) and hormone-sensitive lipase (hsl) were down-regulated. Fatty acid synthase (fas), 6-phosphogluconate dehydro- genase (6pgd) and peroxisome proliferator-activated receptor alpha (pparα) showed highest expression in fish fed intermediate (1.16) DHA/EPA ratio. Overall, this study indicated that dietary DHA/EPA ratio affected fatty acid profiles and significantly influenced lipid metabo- lism including LC-PUFA biosynthesis and other anabolic and catabolic pathways, and also had impacts on antioxidant capacity and hematological characteristics

Molecular Cloning and functional characterization of a putative Elovl4 gene and its expression in response to dietary fatty acid profiles in orange-spotted grouper Epinephelus coioides

Elongase of very long-chain fatty acids (Elovl) 4 probably plays a crucial role in marine fish species, where lack of Elovl2 has been considered as one possible reason for their low long-chain polyunsaturated fatty acids' (LC-PUFAs) biosynthetic capability. Elongase of very long-chain fatty acids 4 is the most recent member of the Elovl family that has been investigated in fish. Here, we report the molecular cloning and functional characterization of putativeelovl4cDNA isolated from marine teleost,Epinephelus coioides, and its expression in response to dietary n-3 LC-PUFA and docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA) ratio. Theelovl4cDNA of grouper was 2341bp including 301bp of 5′-untranslated region (UTR), 918bp of the coding region that encodes 305 amino acids (AA) and 1122bp of 3′UTR. Heterologous expression in yeast demonstrated that grouper Elovl4 could elongate saturated fatty acids (FA), especially 24:0 and 26:0, up to 36:0. Also, grouper Elovl4 effectively converted C20 and C22 polyunsaturated FAs to elongated polyenoic products up to C36. Tissue distribution analysis revealed that Elovl4 were widely transcribed in various tissues with the highest level in eye, brain and testis as described in other teleosts. The transcript level ofelovl4was significantly affected by dietary n-3 LC-PUFA and high LC-PUFA level repressess its expression. However, the ratio of DHA to EPA had no significant influence on its expression. These results may contribute to better understanding the LC-PUFA biosynthetic pathway in this fish species

Functional characterisation of a Fads2 fatty acyl desaturase with delta6/delta8 activity and an Elovl5 with C16, C18 and C20 elongase activity in the anadromous teleost meagre (Argyrosomus regius)

The meagre, Argyrosomus regius, is a carnivorous fish with great potential to diversify finfish aquaculture in the Mediterranean. However, currently nothing is known about their essential fatty acid requirements. Meagres are marine fish but also display anadromous behaviour migrating to river estuaries to spawn, and thus may provide an insight to the influence of diadromy on biosynthetic ability for long-chain polyunsaturated fatty acid (LC-PUFA). Our primary aim was to characterise key cDNAs (fatty acyl desaturases and elongases) of the biosynthetic pathway as a key step to establish the capacity of meagre for LC-PUFA biosynthesis from shorter chain PUFA. The cDNA sequences of a fatty acyl desaturase (Fads) and elongase of very long-chain fatty acids (Elovl) were obtained using PCR-based methodologies, and function of the proteins was investigated by expression of the coding sequences of the putative desaturase and elongase in the yeast Saccharomyces cerevisiae. The tissue distribution of both cDNAs was studied by reverse transcription PCR. Our results demonstrated that meagre possesses at least one fatty acyl desaturase and one elongase involved in the endogenous production of LC-PUFA. The meagre desaturase and elongase were identified as orthologues of Fads2 and Elovl5, respectively. Functionally, the desaturase had dual Δ6/Δ8 activity, whereas the elongase exhibited high elongation efficiency for C18 and C20 PUFA with low activity towards C22 PUFA. However, we also showed that the meagre Elovl5 elongated 16:3n - 3 to 18:3n - 3, the first time that C16 elongation activity had been demonstrated for a fish elongase. Similar to other marine teleosts, expression of fads2 and elovl5 transcripts was highest in brain. The functions and expression of the meagre Fads2 and Elovl5 proteins suggest that the meagre has a ‘marine type' LC-PUFA biosynthetic pathway, and that its anadromous behaviour has no major influence