Aquamax: Sustainable aquafeeds to maximize the health effects of farmed fish for consumers

Jornada de Divulgación Proyecto Europeo AQUAMAX celebrada en el Instituto de Acuicultura de Torre de la Sal (Castellón) el 24 de noviembre de 2010.El Instituto de Acuicultura Torre de la Sal del CSIC organiza una jornada que tiene como objetivo divulgar la investigación sobre el proyecto europeo AQUAMAX. Este proyecto centra su investigación en reemplazar en lo posible los piensos basados en pescado y el aceite de pescado usado actualmente para alimentar a los peces por recursos alternativos libres de contaminantes. Se conseguiría así maximizar el crecimiento, mejorar la conversión del alimento e incrementar la salud y el bienestar de los peces criados en piscifactorías. Además aumentaría la seguridad y calidad del producto que llega finalmente a los consumidores. La jornada contará con la participación del Doctor S. Kaushik del Instituto Nacional para la Investigación Agronómica de Francia, el Doctor Marc Berntssen del Instituto Nacional de Nutrición e Investigación del Alimento Marino de Noruega y el Profesor Jaume Pérez del Instituto de Acuicultura Torre de la Sal del CSIC. En las diferentes intervenciones se tratarán temas del proyecto AQUAMAX como la calidad de los alimentos, la seguridad en la alimentación, así como diversas investigaciones basadas en las doradas.Peer Reviewe

Exploring Uncoupling Proteins and Antioxidant Mechanisms under Acute Cold Exposure in Brains of Fish

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28°C to 18°C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%–90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%–100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18°C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure

A transcriptomic approach to study the effect of long-term starvation and diet composition on the expression of mitochondrial oxidative phosphorylation genes in gilthead sea bream (Sparus aurata)

Abstract Background The impact of nutritional status and diet composition on mitochondrial oxidative phosphorylation (OXPHOS) in fish remains largely unknown. To identify biomarkers of interest in nutritional studies, herein we obtained a deep-coverage transcriptome by 454 pyrosequencing of liver and skeletal muscle cDNA normalised libraries from long-term starved gilthead sea bream (Sparus aurata) and fish fed different diets. Results After clean-up of high-throughput deep sequencing reads, 699,991 and 555,031 high-quality reads allowed de novo assembly of liver and skeletal muscle sequences, respectively (average length: 374 and 441 bp; total megabases: 262 and 245 Mbp). An additional incremental assembly was completed by integrating data from both tissues (hybrid assembly). Assembly of hybrid, liver and skeletal muscle transcriptomes yielded, respectively, 19,530, 11,545 and 10,599 isotigs (average length: 1330, 1208 and 1390 bp, respectively) that were grouped into 15,954, 10,033 and 9189 isogroups. Following annotation, hybrid transcriptomic data were used to construct an oligonucleotide microarray to analyse nutritional regulation of the expression of 129 genes involved in OXPHOS in S. aurata. Starvation upregulated cytochrome c oxidase components and other key OXPHOS genes in the liver, which exhibited higher sensitive to food deprivation than the skeletal muscle. However, diet composition affected OXPHOS in the skeletal muscle to a greater extent than in the liver: most of genes upregulated under starvation presented higher expression among fish fed a high carbohydrate/low protein diet. Conclusions Our findings indicate that the expression of coenzyme Q-binding protein (COQ10), cytochrome c oxidase subunit 6A2 (COX6A2) and ADP/ATP translocase 3 (SLC25A6) in the liver, and cytochrome c oxidase subunit 5B isoform 1 (COX5B1) in the liver and the skeletal muscle, are sensitive markers of the nutritional condition that may be relevant to assess the effect of changes in the feeding regime and diet composition on fish farming

Gene expression survey of mitochondrial uncoupling proteins (UCP1/UCP3) in gilthead sea bream (Sparus aurata L.)

35 p., figuras, tablas y bibliografíaThe aim of this work is to underline the biological significance of mitochondrial uncoupling proteins (UCPs) in ectothermic fish using the gilthead sea bream (Sparus aurata L.) as an experimental model. A contig of 1990 bp in length was recognized as a UCP1 ortholog after initial searches in the gilthead sea bream AQUAFIRST database (www.sigenae.org/aquafirst). Additional searches were performed in skeletal muscle by RT-PCR, and the amplified PCR product was recognized as UCP3 after sequence completion by 5´and 3´RACE. UCP1 expression was mostly detected in liver, whereas UCP3 transcripts were only found in skeletal and cardiac muscle fibers (white skeletal muscle > red skeletal muscle > heart). Specific gene regulation of UCP1 (liver) and UCP3 (white skeletal muscle) was addressed in physiological models of age, seasonal growth and energy-metabolic unbalances. Both the increase in energy demand (stress confinement) and the reduction in energy supply during adaptive cold response in winter down-regulated UCP1 expression. Conversely, transcript levels of UCP3 were higher with age, seasonal fattening and dietary deficiencies in essential fatty acids leading to the increase in fatty acid flux towards the muscle. This close association between UCP1 and UCP3 with the oxidative and metabolic tissue status is perhaps directly related to the ancestral protein UCP function, and allows the use of UCPs as lipotoxicity markers in ectothermic fish.This work was partially funded by the European Union (FOOD-CT-2006-16249: Sustainable Aquafeeds to Maximise the Health Benefits of Farmed Fish for Consumers, AQUAMAX) and Spanish (Ingenio-2010 Programme; Improvement of Aquaculture Production by the use of biotechnological tools, AQUAGENOMICS) projects. AB-N was recipient of a PhD fellowship from CSIC-BANCAJA.Peer reviewe

Tissue-specific gene expression and functional regulation of uncoupling protein 2 (UCP2) by hypoxia and nutrient availability in gilthead sea bream (Sparus aurata): Implications on the physiological significance of UCP1-3 variants

The aim of this study was to assess in an integrative manner the physiological regulation of uncoupling protein 2 (UCP2) in gilthead sea bream. A contig of 1,325 nucleotides in length with an open reading frame of 307 amino acids was recognized as UCP2 after searches in our transcriptome reference database (http://www.nutrigroup-iats.org/seabreamdb). Gene expression mapping by quantitative real-time PCR revealed a ubiquitous profile that clearly differs from that of UCP1 and UCP3 variants with the greatest abundance in liver and white skeletal muscle, respectively. The greatest abundance of UCP2 transcripts was found in the heart, with a relatively high expression level in blood cells, where UCP1 and UCP3 transcripts were practically undetectable. Functional studies revealed that UCP2 mRNA expression remains either unaltered or up-regulated upon feed restriction in glycolytic (white skeletal muscle) and highly oxidative muscle tissues (heart and red skeletal muscle), respectively. In contrast, exposure to hypoxic conditions (18-19 % oxygen saturation) markedly down-regulated the UCP2 mRNA expression in blood cells in a cellular environment with increased haematocrit, blood haemoglobin content, and circulating levels of glucose and lactate, and total plasma antioxidant activity. These findings demonstrated that UCP2 expression is highly regulated at the transcriptional level, arising this UCP variant as an important piece of the complex trade-off between metabolic and redox sensors. This feature would avoid the activation of futile cycles of energy wastage if changes in tissue oxidative and antioxidant metabolic capabilities are able to maintain the production of reactive oxygen species at a low regulated level. © 2013 Springer Science+Business Media Dordrecht.This work was funded by the EU ARRAINA project (Advanced Research Initiatives for Nutrition and Aquaculture, FP7/2007–2013; Grant Agreement No. 288925). Additional funding was obtained from Generalitat Valenciana (Research Grant PROMETEO 2010/006) and the Spanish Government through AQUAGENOMICS (Ingenio-2010 Programme) and AQUAFAT (AGL2009-07797) projects.Peer Reviewe

Unraveling the molecular signatures of oxidative phosphorylation to cope with the nutritionally changing metabolic capabilities of liver and muscle tissues in farmed fish

Mitochondrial oxidative phosphorylation provides over 90% of the energy produced by aerobic organisms, therefore the regulation of mitochondrial activity is a major issue for coping with the changing environment and energy needs. In fish, there is a large body of evidence of adaptive changes in enzymatic activities of the OXPHOS pathway, but less is known at the transcriptional level and the first aim of the present study was to define the molecular identity of the actively transcribed subunits of the mitochondrial respiratory chain of a livestock animal, using gilthead sea bream as a model of farmed fish with a high added value for European aquaculture. Extensive BLAST searches in our transcriptomic database (www.nutrigroup-iats.org/seabreamdb) yielded 97 new sequences with a high coverage of catalytic, regulatory and assembly factors of Complex I to V. This was the basis for the development of a PCR array for the simultaneous profiling of 88 selected genes. This new genomic resource allowed the differential gene expression of liver and muscle tissues in a model of 10 fasting days. A consistent down-regulated response involving 72 genes was made by the liver, whereas an up-regulated response with 29 and 10 differentially expressed genes was found in white skeletal muscle and heart, respectively. This differential regulation was mostly mediated by nuclear-encoded genes (skeletal muscle) or both mitochondrial- and nuclear-encoded genes (liver, heart), which is indicative of a complex and differential regulation of mitochondrial and nuclear genomes, according to the changes in the lipogenic activity of liver and the oxidative capacity of glycolytic and highly oxidative muscle tissues. These insights contribute to the identification of the most responsive elements of OXPHOS in each tissue, which is of relevance for the appropriate gene targeting of nutritional and/or environmental metabolic disturbances in livestock animals. © 2015 Bermejo-Nogales et al.This work was funded by the EU AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research, FP7/2007/2013; grant agreement No. 262336; www.aquaexcel.eu) project. Additional funding was received by Generalitat Valenciana (PROMETEOII/2014/085; www.iats.csic.es/nisaam) and from the Spanish Government through AQUAGENOMICS (Ingenio-2010 Programme; www.aquagenomics.es) project.Peer Reviewe

Respiration uncoupling in gilthead sea bream

25 p., 2 figures, 2 tables and referencesThe physiological regulation of the mitochondrial uncoupling protein 3 (UCP3) remains practically unexplored in fish and the aim of this study was to examine the effects of ration size on the regulation of UCP3 in heart, red skeletal muscle and white skeletal muscle of gilthead sea bream (Sparus aurata L.). Juvenile fish were fed at three different levels for 11weeks: i) full ration until visual satiety (R 100 group), ii) 70% of satiation (R 70 group) and iii) 70% of satiation with two finishing weeks at the maintenance ration (20% of the satiation level) (R 70-20 group). The thirty percent feed restriction improved fish performance, increasing feed conversion efficiency and circulating levels of insulin-like growth factor-I (IGF-I). Fish of the R 70-20 group showed reduced growth and low circulating levels of IGF-I in combination with increased circulating concentrations of growth hormone and free fatty acids. Feed restriction did not alter UCP3 transcript levels in white skeletal muscle, but improved this tissue's oxidative capacity as assessed by changes in glycolytic and oxidative mitochondrial enzyme activities. In contrast, in cardiac and red skeletal muscle tissues, this dietary treatment primarily increased UCP3 mRNA expression. The respiratory control ratio of freshly isolated heart mitochondria was slightly lower in R 70-20 fish than in R 100 fish, which suggests that there was an increase in mitochondrial uncoupling concomitant with the enhanced UCP3 mRNA expression. Altogether, these findings highlight the different adaptive mechanism of glycolytic and highly oxidative muscle tissues for their rapid adjustment to varying feed intake.The research was funded by AQUAGENOMICS (CSD2007-00002, Improvement of aquaculture production by the use of biotechnological tools,) and AQUAFAT (AGL2009-07797; Predictive modeling of flesh fatty acid composition in cultured fish species with different muscle lipid content) projects. Additional funding was obtained from the “Generalitat Valenciana” (research grant PROMETEO 2010/006).Peer reviewe

Gene expression profiling of whole blood cells supports a more efficient mitochondrial respiration in hypoxia-challenged gilthead sea bream (Sparus aurata)

[Background] Acclimation to abiotic challenges, including decreases in O2 availability, requires physiological and anatomical phenotyping to accommodate the organism to the environmental conditions. The retention of a nucleus and functional mitochondria in mature fish red blood cells makes blood a promising tissue to analyse the transcriptome and metabolic responses of hypoxia-challenged fish in an integrative and non-invasive manner.[Methods] Juvenile gilthead sea bream (Sparus aurata) were reared at 20–21 °C under normoxic conditions (> 85% O2 saturation) followed by exposure to a gradual decrease in water O2 concentration to 3.0 ppm (41–42% O2 saturation) for 24 h or 1.3 ppm (18–19% O2 saturation) for up to 4 h. Blood samples were collected at three different sampling points for haematological, biochemical and transcriptomic analysis.[Results] Blood physiological hallmarks remained almost unaltered at 3.0 ppm, but the haematocrit and circulating levels of haemoglobin, glucose and lactate were consistently increased when fish were maintained below the limiting oxygen saturation at 1.3 ppm. These findings were concurrent with an increase in total plasma antioxidant activity and plasma cortisol levels, whereas the opposite trend was observed for growth-promoting factors, such as insulin-like growth factor I. Additionally, gene expression profiling of whole blood cells revealed changes in upstream master regulators of mitochondria (pgcβ and nrf1), antioxidant enzymes (gpx1, gst3, and sod2), outer and inner membrane translocases (tom70, tom22, tim44, tim10, and tim9), components of the mitochondrial dynamics system (mfn2, miffb, miro1a, and miro2), apoptotic factors (aifm1), uncoupling proteins (ucp2) and oxidative enzymes of fatty acid β-oxidation (acca2, ech, and hadh), the tricarboxylic acid cycle (cs) and the oxidative phosphorylation pathway. The overall response is an extensive reduction in gene expression of almost all respiratory chain enzyme subunits of the five complexes, although mitochondrial-encoded catalytic subunits and nuclear-encoded regulatory subunits of Complex IV were primarily increased in hypoxic fish.[Conclusions] Our results demonstrate the re-adjustment of mitochondrial machinery at transcriptional level to cope with a decreased basal metabolic rate, consistent with a low risk of oxidative stress, diminished aerobic ATP production and higher O2-carrying capacity. Taken together, these results suggest that whole blood cells can be used as a highly informative target tissue of metabolic condition.This work was financially supported by a grant from the European Commission of the European Union under the Horizon 2020 research infrastructure project AQUAEXCEL2020 (652831). Additional funding was provided by Generalitat Valenciana (PROMETEOII/2014/085). JAMS received a Postdoctoral Research Fellowship (Juan de la Cierva-Formación, Reference FJCI-2014-20,161) from MINECO.Peer reviewe