Lipid classes and their content of n-3 highly unsaturated fatty acids (HUFA) in <i>Artemia franciscana</i> after hatching, HUFA-enrichment and subsequent starvation

The distribution of n-3 highly unsaturated fatty acids (HUFA) over the major neutral and polar lipid classes was determined for two predominant types of live food used in the larviculture of marine fish and shrimp, i.e. freshly hatched and HUFA-enriched Artemia, and compared with data reported in the literature for wild copepods, representing the natural diet of these larvae. Lipid class composition and their content of n-3 HUFA, particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), were assessed in freshly hatched, HUFA-enriched and subsequently starved Artemia franciscana. The n-3 HUFA enrichment was based on feeding Artemia a lipid emulsion in which either fatty acid ethyl esters (EE, diluted with olive oil) or triacylglycerol (TAG) provided a level of 30% n-3 HUFA. Enrichment of Artemia with either type of the lipid emulsions resulted in an increase of total lipid content from 20.0 to 28.2-28.7% of dry matter mainly due to the accumulation of neutral lipid, primarily TAG (from 82 to 158 mg g(-1) dry wt in freshly hatched and 24-h enriched Artemia). Enriched brine shrimp utilized up to 27-30% of their TAG content during 72 h of starvation at 12 degrees C. The absolute tissue concentrations of polar lipids remained constant at 71 to 79 mg g(-1) dry wt throughout the enrichment and subsequent starvation. The level of n-3 HUFA increased drastically during enrichment from 6.3% of total fatty acids (8.2 mg g(-1) dry wt) in freshly hatched nauplii to between 20.4 and 21.8% (40.4 to 43.2 mg g(-1) dry wt) in 24-h enriched Artemia and was not significantly affected by the source of n-3 HUFA. During starvation, 18:0, 20:4n-6 and 20:5n-3 were retained, whereas 18:4n-3, 22:5n-3 and 22:6n-3 were specifically catabolized. The major polar lipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), of freshly hatched Artemia showed very low levels of DHA ( 64% of the EPA and > 91% of the total DHA present. This is in sharp contrast with the high levels of n-3 HUFA, in particular DHA, in the polar lipid fraction reported for wild copepods. The contrasting distribution of DHA in the neutral and polar lipid fractions of enriched brine shrimp compared to the natural diet may influence the efficacy of this essential fatty acid for marine fish larvae in aquaculture systems

Tissue distribution of lipid and fatty acid metabolism and transcription factors genes in adult Atlantic bluefin tuna (Thunnus thynnus L.)

To determine expression of the major lipid pathways in tissues of adult bluefin tuna 8 individuals were used for collecting samples for tissue expression of key lipid metabolism genes. Triplicate sets of samples of brain, gills, heart, kidney, spleen, liver, intestine, white muscle, red muscle, adipose tissue ovary and testis were collected. Expression of genes was determined by qPCR. Tissue expression profiles showed that PUFA biosynthetic pathway genes were expressed in all tissues examined, highest expression in brain, liver and testis. Elongase elovl5 showed higher expression than desaturase fads2d6 in all other tissues, with low expression of in red muscle and ovaries. Transcription factors, pparα and pparγ showed parallel expression, with adipose tissue with the highest relative copy number, followed by intestine>testis>liver. The expression of lxr was low in liver, with highest expression in testis, brain and kidney. Similarly, rxr was poorly expressed in liver with higher expression in muscle, spleen and brain. The rank order of expression of srebp1 was brain, testis, ovary, intestines, kidney, gill, liver, white muscle, spleen, heart and red muscle. For srebp2 the highest expression was shown in brain, testis and adipose with lowest expression in heart and white muscle. Expression of fabp2 was highest in intestine, brain and heart with lower levels in liver, red muscle, adipose and kidney. fabp4 showed highest expression in ovaries with liver showing the lowest. fabp7 showed highest expression levels in brain and testis with lowest values in liver. cptI expression was highest in brain and lowest in liver. Similarly, the relative copy number for fas was highest in brain followed by gonads, gill and liver, with white muscle showing lowest expression. The expression of aco was highest in adipose and intestine, followed by liver, kidney and brain. The expression of hmgcl was highest in ovary followed by adipose, brain and testis, with lowest expression in liver. Expression of lpl was highest in testis and lowest in ovary, with liver and white muscle, adipose, heart, gills and red muscle, kidney, intestine showing intermediate levels of expression. This research was supported by projects from the Junta de Andalucía, Proyecto de Excelencia de Promoción General del Conocimiento Ref. RNM 0733, and Programa Estatal de Investigación del Ministerio de Economía y Competitividad Ref. AGL2014-52003-C2-1-R. AQUACULTURE, ENDOCRINOLOGY AND TOXICOLOGY 218. VI IBERIAN CONGRESS OF ICHTHYOLOGY MURCIA, SPAIN 21st · 24th June P-04

Molecular aspects of lipid metabolism, digestibility and antioxidant status of Atlantic bluefin tuna (T. thynnus L.) larvae during first feeding

Atlantic bluefin tuna (Thunnus thynnus L.; ABT) larvae were fed on enriched rotifers Brachionus rotundiformis and copepod nauplii Acartia tonsa from first feeding to 15 days post hatching. Rotifers were enriched with five different commercial products: OG, MG, AG and RA plus selenium and vitamin E. Copepods (COP) were cultured with the algae Rhodomonas baltica. Metabolic processes were studied by determining the expression of 30 genes related to lipid metabolism (transcription factors, fatty acid metabolism and lipid homeostasis), antioxidant enzymes, myogenesis and digestive enzymes. Growth and development parameters and high expression of myogenesis genes myhc2 and tropo indicated that COP were better than enriched rotifers as live prey for first feeding ABT. COP and AG-fed larvae showed the lowest values for the transcription factors pparã and srebp2. The expression of fas showed differences among treatments, with highest relative expression in COP-fed larvae and those fed with RA rotifers. In relation to fatty acid catabolism, larvae fed RA had the highest aco expression levels, with the lowest observed in those fed COP. The expression profiles of lipid homeostasis genes showed that larvae fed COP had higher fabp2 and 4 expressions. Larvae fed AG showed the lowest lpl expression levels, with highest values observed in larvae fed OG. Regarding antioxidant enzyme gene expression, sod showed highest values in larvae fed COP and RA, with larvae fed MG rotifers showing lowest expression levels. A similar pattern was observed for the expression of cat and gpx1 and 4. The expression of genes for digestive enzymes showed that tryp expression levels were highest in COP-fed larvae but, in contrast, COP-fed larvae showed the lowest anpep and alp levels. ABT larvae fed AG displayed the lowest expression level of pla2. bal1 and bal2 presented similar expression patterns, with highest values in COP-fed ABT and lowest expression in larvae fed AG rotifers. Copepods were a superior live prey for first feeding ABT larvae compared to enriched rotifers, as indicated by the higher growth and flexion index achieved by COP-fed larvae, possibly reflecting the higher protein content of the copepods.This work was supported by the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía, Proyecto de Excelencia de Promoción General del Conocimiento [Ref. RNM 733, 2012], and Programa Estatal de Investigación del Ministerio de Economía y Competitividad [Ref. AGL2014-52003-C2-1-R, 2014].Versión del edito

COPEPODS OR ROTIFERS? EVALUATING THE USE OF DIFFERENT FEEDING PROTOCOLS FOR LARVAE OF ATLANTIC BLUEFIN TUNA (Thunnus thynnus. L)

There are still many issues that require to be solved in larval rearing of Atlantic bluefin tuna (Thunnus thynnus; ABT) to prevent “mass-mortality” during this developmental stage. Initial data related to the feeding sequence of ABT larvae suggested that mortality observed during the first stages of life could be due partly to nutritional deficiencies. Previous studies demonstrated that copepods appeared to be a superior live prey compared to rotifers during the first two weeks of life. Our overarching aim was to evaluate different feeding strategies during first feeding of ABT larvae from a performance, compositional and molecular perspective. In order to do so, two groups of ABT larvae were fed with either copepod (Acartia tonsa; C) nauplii or rotifers (Brachionus rotundiformis; R) enriched with Algamac 3050® from mouth opening to 13 days after hatching (dah). After this, the group C-larvae was fed either Artemia enriched with Algamac 3050® (CA), Acartia nauplii and copepodites (CC) or sea bream (Sparus aurata) yolk-sac larvae (CY), while the R group passed on to being fed on Artermia enriched with Algamac 3050® (RA) up to 18 dah. After 13 dah, larvae fed C grew more than those fed R although there were no differences in survival. ABT larvae fed R accumulated highest eicosapentaenoate (EPA) but lowest docosahexaenoate (DHA) and total n-3 long-chain polyunsaturated fatty acids (LC-PUFA) than C-fed larvae, reflecting dietary contents. Indeed, there was no activation in the expression of the enzymes involved in LC-PUFA biosynthesis. However, the different live prey elicited regulation of transcription factor, digestive enzyme, lipid metabolism and oxidative stress genes. At 18 dah larvae fed CY and CA were the largest size with larvae fed RA displaying the lowest growth with no differences in survival among the dietary treatments. The highest DHA contents were found in ABT larvae fed CC and CY, whereas the lowest contents were found in RA-fed larvae. Indeed, RA-fed larvae showed the highest level of the intermediate product n-3 docosapentaenoate, which could be reflecting up-regulation in the biosynthetic pathway although this was not supported by gene expression data

Development of feeds for juvenile Atlantic bluefin tuna (Thunnus thynnus, L): effect of lipid level and source

In conclusion, the present study suggests that ABT juveniles can be grown on inert extruded dry feeds that result in good fish growth and accumulation of the health-promoting fatty acid DHA. Furthermore, a blend of VO and KO could be used as the dietary lipid source up to a dietary lipid level of 15 % without affecting fish performance. The expression of lipid metabolism genes in ABT liver showed a different response to dietary lipid level/fatty acid profile, consistent with previous data indicating limited n-3 LC-PUFA biosynthetic capability in ABT. However, gene expression showed some differences between the two trials, which highlight how the genetic background of different batches of ABT juveniles could affect the regulation of metabolic gene expression and thus be a factor in weaning success. The expression of antioxidant enzymes was also altered by diet, related to dietary contents of antioxidant nutrients. Thus, further studies are required in order to fully elucidate the lipid and fatty acid requirements of this iconic species in relation to dietary sources and production cost

Performance, feed utilization and hepatic molecular metabolic response of weaned juvenile Atlantic bluefin tuna (Thunnus thynnus, L): effect of lipid level and source

The development of formulated diets and feeds is essential to increase production of farmed tuna species. There is limited knowledge of this topic, mainly on Pacific Bluefin tuna (Thunnus orientalis) in Japan, whereas no major attempts have been made with Atlantic Bluefin tuna (Thunnus thynnus; ABT). In the present study, two trials were performed using inert formulated diets as on-growing feeds for weaned ABT juvenile in order to establish adequate dietary levels of both lipid and omega-3 long-chain polyunsaturated fatty acids (LC-PUFA). In a first trial, ABT (initial weight = 2.9±0.9g) were fed for 10 days with either a commercial (Magokoro®, MGK) or two experimental feeds with two different lipid levels (15 or 20%) using krill oil (KO) as the single lipid source in order to estimate the suitable lipid content. Fish fed MGK displayed the highest growth, followed by 15KO, with no differences in fish survival. Thus, a lipid content of 15% was considered better than 20% for ABT juveniles. In the second trial, fish (initial weight = 3.3 ± 0.6g) were fed either MGK, 15KO or a feed containing 15% lipid with a combination (1:1, v/v) KO and rapeseed oil (RO) (15KORO). Fish fed 15KO and 15KORO showed the highest growth in terms of weight and fork length (including weight gain and SGR). Increasing dietary lipid level or adding RO to the feeds did not increase liver lipid content. The liver fatty acid profile largely reflected dietary intake confirming very limited LC-PUFA biosynthetic activity for this teleost species. In this respect, liver of fish fed 15KO and 20KO displayed the highest contents of docosahexaenoic acid (DHA). The hepatic expression of genes of lipid and fatty acid metabolism, transcription factors, and antioxidant enzymes was investigated with many of the genes showing regulation by both dietary lipid and LC-PUFA contents. The present study showed promising results that suggested ABT juveniles can be on grown on inert dry feeds that supported good fish growth and the accumulation of the health-promoting fatty acid DHA. Further studies are required in order to fully elucidate lipid and fatty acid requirements of this iconic species regarding dietary sources and production costs

Taurine metabolism and effects of inclusion levels in rotifer (Brachionus rotundiformis, Tschugunoff, 1921) on Atlantic bluefin tuna (Thunnus thynnus, L.) larvae

Taurine appears to be a crucial nutrient for teleosts, especially top predator species such as Atlantic bluefin tuna (Thunnus thynnus, L.; ABT). While dietary taurine supplementation has been highly recommended, there is a lack of studies on taurine assimilation and biosynthesis for this iconic species. The present study aims to provide insight into the molecular mechanisms involved in taurine biosynthesis and transport in ABT by studying tissue distribution and ontogenetic development of expression of cysteine dioxygenase (cdo), cysteine sulfinic acid decarboxylase (csad), 2-aminoethanethiol dioxygenase (ado) and taurine transporter (tauT) in response to graded levels of dietary taurine supplementation. The full open reading frame (ORF) for cdo and partial sequences for csad, ado and tauT were obtained, with the translated polypeptides being 202, 176, 166 and 324 amino acids, respectively. All three showed characteristics such as cupin motifs in Cdo and predicted N-glycosylation sites in Taut that are common to these genes in other species. Phylogenetic analysis showed that the ABT sequences clustered with sequences of other teleosts, and separately from mammals and molluscs. Tissue distribution varied, with adipose tissue, kidney, white muscle and testis/brain showing highest expression of cdo, csad, ado and tauT, respectively. Whole larvae expression of csad peaked at 15 dah, whereas the other genes generally increased throughout development to show highest expression at 25 dah. The nutritional trial was carried out by feeding ABT larvae from mouth opening to 14 days after hatching (dah) with rotifers (Brachionus rotundiformis) enriched with 4 different levels of taurine: 0.0 (tau0), 0.5 (tau0.5), 1.0 (tau1), and 2.0 g taurine per 106 rotifers (tau2). Rotifers effectively accumulated taurine with ABT larvae fed on treatment tau2 attaining the highest concentration of taurine. However, ABT larvae fed tau1 displayed higher growth and survival, and flexion index at 14 dah, than larvae fed the other taurine levels. Larvae fed tau1 also showed generally higher expression of tauT and cdo and digestive and antioxidant enzyme genes. While this study showed that larval ABT express taurine metabolism genes, suggesting possible synthesis that could contribute to the taurine pool in the fish, larval performance was enhanced by a level of dietary taurine (3.7 mg taurine g−1 rotifer) supplied by enrichment of rotifers at 1 g taurine per 106 rotifers.En prensa2,04

ATLANTIC BLUEFIN TUNA (Thunnus thynnus, L.) LARVAE ANTIOXIDANT MOLECULAR FUNCTIONS INDUCED BY DIETARY SELENIUM IN ROTIFER Brachionus rotundiformis

including iodothyronine deiodinases 1, 2 and 3 (dio1, dio2, dio3) was unaffected. Feeding Se enriched rotifers effectively increased Se in ABT larvae tissue. The improved growth observed in Se supplemented treatments might be related to an accelerated development as the flexion index was significantly higher in all Se enriched treatments compared to the non-supplemented control. A similar effect by Se supplementation has been previously described in Senegalese sole (Solea senegalensis) in relation to an enhanced thyroid hormone activity by Se supplementation (Ribeiro et al., 2012). The Se level of 0.10 µg g-1 dw measured in non-supplemented rotifers is below known requirements in fish (Antony Jesu Prabhu et al. 2016). In contrast, rotifers supplemented with the lowest Se level (Se3) contained 4.42 µg Se g-1, which might be sufficient to cover requirements for this mineral as selenoproteins displayed maximum expression in ABT larvae fed this treatment. The increased seleno-enzyme production might have contributed towards an improved antioxidant status in ABT larvae, indicated by a transcriptional downregulation of redox sensitive antioxidant enzymes cat and sod. In conclusion, rotifers without Se enrichment are suboptimal for ABT larvae at first feeding. A dietary Se level of 4.42 µg g-1 dw is recommended as it boosted growth performance and improved the antioxidant status in ABT larvae

Central and peripheral clocks in Atlantic bluefin tuna (Thunnus thynnus, L.): Daily rhythmicity of hepatic lipid metabolism and digestive genes

Atlantic bluefin tuna (ABT; Thunnus thynnus) is a highly regarded and consumed species, but farming is still in its infancy. Currently, nothing is known about the presence of circadian rhythmicity at central or peripheral tissues, or if there are daily rhythms in expression of genes involved in lipid metabolism. In order to elucidate clock gene regulation of genes of lipid metabolism in ABT, six clock genes (bmal1, clock, cry1, cry2, per1 and per2) were sequenced and 24 h expression of these genes determined in brain and liver of fish acclimated to a light:dark (L:D) photoperiod. Additionally, the daily expression of lipid metabolism and digestive enzyme genes in ABT was also determined in liver. All six clock genes displayed rhythmicity in the brain and liver, other than cry2, which did not show an acrophase in liver. In liver, all the transcription factors analysed other than srebp1 and srebp2 displayed rhythmicity, with lxr and pparα displaying diurnal expression, whereas pparγ was highly expressed at the end of the scotophase. Some of the target genes of lxr such as elovl5 and lpl also oscillated rhythmically, with acrophases during the photophase. In contrast, only three of the eight digestive enzyme genes studied displayed rhythmicity, at different times of the day, suggesting that either ABT display different feeding periods or the digestion of some nutrients (e.g. lipids) is prioritized over others. The present study showed that clock and lipid metabolism genes displayed strong daily rhythmicity in ABT brain and liver, which could be an area of considerable interest for the establishment of efficient feeding protocols in this new aquaculture species.En prensa2,04

Molecular and functional characterisation of a putative elovl4 gene and its expression in response to dietary fatty acid profile in Atlantic bluefin tuna (Thunnus thynnus)

Elongation of very long-chain fatty acid 4 (Elovl4) proteins are involved in the biosynthesis of very long-chain (> C24) fatty acids and in many teleost fish species they are key enzymes in the pathway for the production of docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Therefore, Elovl4 may be particularly important in Atlantic bluefin tuna (ABT; Thunnus thynnus) characterised by having high DHA to EPA ratios. The present study cloned and characterised both the function and expression of an elovl4 cDNA from ABT. The Elovl4 had an open reading frame of 915 base pairs encoding a putative protein of 304 amino acids. Alignment and phylogenetic analyses indicated that the Elovl4 isoform identified in the present study was an Elovl4b. Functional characterisation demonstrated that the Elovl4b enzyme had elongase activity towards all the polyunsaturated fatty acid (PUFA) substrates assayed. The ABT Elovl4b contributed to DHA biosynthesis by elongation of EPA and DPA to 24:5n-3, the latter being desaturated to 24:6n-3 by the action of fads2 (Δ6 desaturase). Additionally, the ABT Elovl4b has a role in the biosynthesis of very long-chain PUFA up to C34, compounds of key structural roles in neural tissues such as eye and brain, which had high levels of elovl4b transcripts. Surprisingly, while the relative expression of fads2, required for the production of DHA from EPA, was increased in liver of ABT fed a diet with reduced levels of EPA and DHA, expression of elovl4b was reduced. Results indicated that ABT has enzymes necessary for endogenous production of DHA from EPA and demonstrate that Elovl4b can effectively compensate for absence of Elovl2.Versión del edito