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Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection., Fundación para la Ciencia y la Tecnología (FECYT), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection., Fundación para la Ciencia y la Tecnología (FECYT), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)- box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction. © 2013 CSIRO.

In order to investigate the effects of dietary protein hydrolysates (PH) on larval growth performance, skeleton quality and proteome expression, triplicate groups of white seabream (Diplodus sargus) larvae were co-fed from first-feeding with live feed and three microencapsulated diets differing in the molecular weight of their PH fraction (Control – inclusion of CPSP-90; H – inclusion of a high amount in 0.5–30 kDa hydrolysates; L – inclusion of a high amount in <0.5 kDa hydrolysates). At 15 days after hatching (DAH), proteome expression changes were assessed in entire larvae by two-dimensional gel electrophoresis and the quality of larval skeleton was analysed at 28 DAH through double staining of cartilage and bone. Dietary PH fractions tested affected growth, the larvae fed diet L being significantly larger than those fed diet H, but it did not affect the incidence of deformed larvae, nor the number of deformities per fish. Two-dimensional analysis of larvae proteome allowed the detection and the comparative quantification of a total of 709 protein spots having a pI between 4 and 7, around half of which had an expression significantly affected by dietary treatment, the main difference being between proteome of Control larvae with those of both groups L and H. From these spots, 52 proteins involved in diverse processes such as cytoskeletal dynamics, energetic, lipoprotein, amino acid (AA), and nucleotide metabolisms, protein chaperoning and degradation, and signal transduction, were identified. This study revealed that the molecular weight of the dietary protein hydrolysate fraction had a minor impact on skeletal deformities in white seabream larvae, but affected growth performance and had a strong impact on larvae whole body proteome.

Two different modes for regulation of stomach acid secretion have been described in vertebrates. Some species exhibit a continuous acid secretion maintaining a low gastric pH during fasting. Others, as some teleosts, maintain a neutral gastric pH during fasting while the hydrochloric acid is released only after the ingestion of a meal. Those different patterns seem to be closely related to specific feeding habits. However, our recent observations suggest that this acidification pattern could be modified by changes in daily feeding frequency and time schedule. The aim of this study was to advance in understanding the regulation mechanisms of stomach digestion and pattern of acid secretion in teleost fish. We have examined the postprandial pattern of gastric pH, pepsin activity, and mRNA expression for pepsinogen and proton pump in white seabream juveniles maintained under a light/dark 12/12 hours cycle and receiving only one morning meal. The pepsin activity was analyzed according to the standard protocol buffering at pH 2 and using the actual pH measured in the stomach. The results show how the enzyme precursor is permanently available while the hydrochloric acid, which activates the zymogen fraction, is secreted just after the ingestion of food. Results also reveal that analytical protocol at pH 2 notably overestimates true pepsin activity in fish stomach. The expression of the mRNA encoding pepsinogen and proton pump exhibited almost parallel patterns, with notable increases during the darkness period and sharp decreases just before the morning meal. These results indicate that white seabream uses the resting hours for recovering the mRNA stock that will be quickly used during the feeding process. Our data clearly shows that both daily illumination pattern and feeding time are involved at different level in the regulation of the secretion of digestive juices.

Currently, several species of grouper are already being produced in Asia for commercial purposes. Several individuals are kept as broodstock in research facilities throughout the Mediterranean.

Low sperm volume and quality is pointed out as a major reason for the low fertilization rates observed in Senegalese sole (Solea senegalensis) broodstocks both wild and F1. R ecent studies by our group have shown that the poor and variable sperm quality is related to the high sensibility of plasma membrane to seawater during fertilization. Diet enrichment in fatty acids and cholesterol could change the composition of the sperm membrane and the addition of antioxidants would protect sperm to peroxidation.Therefore, in the present work we tested specific diets enriched with DHA, selenium and Vit. E, aiming to improve sperm sole quality.

Cryopreservation of testicular germ cells offers a tool for the conservation and production of species with potential for aquaculture, due to the capacity of spermatogonia differentiating into gametes. These cellshave the ability to proliferate in a host individual after transplantation producing, in their gonad, functionalgametes depending on the host sex determination (Yoshizaki et al., 2011). Cryopreservation andtransplantation of testicular germ cells have been attempted in several mammalian species (Redden et al., 2009) and more recently in some fish (Takeuchi et al . 2009; Yoshizaki et al., 2011). In the present studycryopreservation protocols were developed for testicular germ cells from Senegalese sole ( Solea senegalensis ), a species with several reproductive problems, with the aim to transplant into newly hatched larvae and testtheir efficiency.

The effect of the moon light cycle on plasma melatonin rhythms was examined in Senegal sole (Solea senegalensis) exposed to natural outdoor or artificial indoor lighting conditions. Furthermore, in a second experiment, the effect of the lunar cycle on vitellogenin and sex steroids (Testosterone, T; Estradiol, E2; 11-ketotestosterone, 11kt) was studied using mature individuals during reproductive season. In the first experiment, during full moon, plasma melatonin peaked at night in covered tanks (deprived of night illumination) from both outdoor (133.2 ± 12.8 pg ml-1) and indoor (190.6 ± 41.5 pg ml-1) groups. However, for fish in the open tanks, exposed to approximately 0.3 lux of illumination, nocturnal plasma melatonin was significantly reduced (p<0.05), approaching to mid-light (ML) values, (79.6 ± 7.1 and 81.8 ± 14.0 pg ml-1, for outdoor and indoor groups, respectively). During new moon a similar pattern was observed in outdoor group: fish in the covered tank showed higher melatonin values than those in the open tank, which were exposed to the near undetectable night illumination. In the second experiment, plasma sex steroid concentrations were significantly higher during the full moon compared to the new moon. In the case of females, E2 concentration reduced from 2.4 ± 0.6 to 0.4 ± 0.1 ng ml-1 between full and new moon samplings, while T decreased from 0.3 ± 0.0 to 0.2 ± 0.0 ng ml-1. Vitellogenin, however, did not show such differences between moon phases. In males, 11kt exhibited a plasma concentration of 14.3 ± 2.1 ng ml-1 during full moon and 4.7 ± 0.7 ng ml-1 during new moon while T values were 2.6 ± 0.4 and 1.0 ± 0.1 ng ml-1 for full and new moon, respectively. In conclusion, these findings pointed out the high sensitivity to moon light of the Senegal sole, which could be using the melatonin signalling to synchronize their reproduction rhythms to the lunar cycle.

Earthen ponds are the main production system for seabass and seabream in Portugal and in Southern Spain, in particular in Cádiz province. Different farms use various levels of stocking densities and pond sizes, but in general these are semi-intensive systems covering large areas with ponds ranging from one to several hectares and production levels from 0.5 to 6 Kg/m3 (mostly below 2 kg/m3) at the end of the production cycle.

Earth ponds are the main production system for seabass and seabream in Portugal and in Spain, in particular in the Cádiz province. Different farms use various levels of intensification and pond size, but in general these are semi-intensive ponds covering large areas, ranging from one to several hectares, and with production ranging from 0.5 to 1.5 Kg/m2 at the end of the production cycle. Production costs in this farming system are higher compared to intensive cage farms, and its economic sustainability depends on product differentiation and optimization of production.

Earthen ponds are the main production system for seabass and seabream in Portugal and in Southern Spain, in particular in Cádiz province. Different farms use various levels of stocking densities and pond sizes, but in general these are semi-intensive systems covering large areas with ponds ranging from one to several hectares and production levels from 0.5 to 6 Kg/m3 (mostly below 2 kg/m3) at the end of the production cycle. Although seabass and seabream are traditionally the target species produced in such ponds, there is commonly natural recruitment of wild larvae of other fish species, including Senegalese sole. Previous attempts at ongrowing sole in ponds with seabass and seabream as added value gave varied results in terms of number of individuals recovered, but sole growth rates were promising. Polyculture of seabass and seabream is already an established practice, with proportions of 4:1 normally used. In seabass dominated ponds seabream is used to control growth of macroalgae and to clean the ponds. When seabream production dominates, the carnivorous nature of seabass is used to control populations of smaller fish which enter the ponds naturally and might compete for feed. Polyculture of species from different trophic levels has also been considered an efficient and environmentally sound strategy to minimise the impacts of aquaculture systems, since an important fraction of dissolved nutrients and organic matter is recycled within the pond (Buschmann et al. 1996, Sorgeloos 2001, World Bank 2006).

The European project Sustainable extensive and semi-intensive coastal aquaculture in Southern Europe (SEACASE) included the proposal of 6 Codes of Conduct for the aquaculture systems studied during the project work. These case studies are typical of the countries involved, namely semi-intensive polyculture (Portugal and Spain), extensive polyculture in esteros (Spain), integrated systems (France), valliculture (Italy), nursery ponds (Greece, Portugal, France and Italy) and eel ponds (France). This article was written having as objective the providing of a concise summary of the characteristics that SEACASE collaborators found to be central to build a complete and trustable Certification system for products of sustainable non-intensive aquaculture (for “certification of sustainability”, see Ojeda, 2008). The 6 Codes of Conduct are the basic information to build such a system in the future.

Phenylalanine is the precursor of tyrosine, which is involved in the synthesis of several molecules with key roles in the regulation of metabolism and growth, stress response and pigmentation. In this study, three experimental diets were tested: an amino acid (AA) balanced diet supplemented with phenylalanine, another supplemented with phenylalanine and tyrosine and a non-supplemented AA balanced diet. Rotifers were enriched with liposomes encapsulating free AA in order to obtain a balanced AA profile. The experimental diets resulted in similar larval survival, growth, enzyme activities of AA catabolism and nitrogen excretion in all treatments. High levels of skeletal deformities were registered and significant differences were found between the control and the phenylalanine treatment for the percentage of vertebral compressions in the trunk region of the vertebral column (30% in the control and 5% in the phenylalanine group). A significantly higher survival to a temperature stress test was found for larvae fed the diet supplemented with phenylalanine and tyrosine. The results suggest that supplementation of phenylalanine/tyrosine in fish diets may be useful in order to reduce skeletal deformities and mortalities caused by stress. The present study confirms that AA requirements may be sufficient for covering growth and survival but insufficient to cover other metabolic processes.

Amino acids are the building blocks for growth and the major energy source during fish larval stages. Deficient amino acids can be supplemented in the diets, overcoming problems such as low growth rates and skeletal deformities. In this study, three experimental diets were tested: a balance diet supplemented with lysine, a balance diet supplemented with tryptophan and a control with no supplementation. Trials were conducted with Diplodus sargus larvae from 1 to 25 days after hatching (DAH). A microencapsulated diet was introduced at 15 DAH in co-feeding with live feed and from 20 DAH larvae were fed only this diet. The effect of the supplemented diets was assessed in terms of survival, growth rate, skeletal deformities, ammonia excretion and activity of amino acid catabolism enzymes. The results showed a similar survival in all treatments. However, larvae given tryptophan supplementation had a lower weight on 25 DAH. No significant differences were found in ammonia excretion, frequency or type of deformities or enzymatic activity. Tryptophan and lysine supplementation failed to improve larval growth, survival or larval quality.

Fish larvae present high amino acid requirements due to their high growth rate. Maximizing this growth rate depends on providing a balanced amino acid diet which can fulfil larval amino acid nutritional needs. In this study, two experimental microencapsulated casein diets were tested: one presenting a balanced amino acid profile and another presenting an unbalanced amino acid profile. A control diet, live feed based, was also tested. Trials were performed with larvae from 1 to 25 days after hatching (DAH). Microencapsulated diets were introduced at 8 DAH in co-feeding with live feed and at 15 DAH larvae were fed the microencapsulated diets alone. Results showed a higher survival for the control group (8.6 ± 1.3% versus 4.2 ± 0.6% and 3.2 ± 1.8%) although dry weight and growth were similar in all treatments. The proportion of deformed larvae as well as the ammonia excretion was lower in the group fed a balanced diet than in the unbalanced or control groups (38.3% deformed larvae in control, 30% in larvae fed unbalanced diet and 20% on balanced diet group). Furthermore, larvae fed the microencapsulated diets presented higher docosahexaenoic acid and arachidonic acid levels. This study demonstrates that dietary amino acid profile may play an important role in larval quality. It also shows that balanced microencapsulated diets may improve some of the performance criteria, such as skeletal deformities, compared to live feeds.

A key to success in the culture of marine fish species is the mass production of high quality fry, a process largely dependent on successful first feeding and normal development and growth of fish larvae. In this regard it is important to examine the structural and functional development of the endocrine system (pituitary, thyroid, interrenal glands) during early ontogeny of marine fish. Particularly, the thyroid hormones, thyroxine (T4) and triiodothyronine (T3), influence numerous metabolic processes, such as growth, differentiation, metamorphosis, reproduction, respiration, migratory behaviour, central nervous system activity, seasonal adaptation, etc. Therefore the aim of this study was to describe the development of the thyroid gland and the ontogeny appearance of the thyroid hormones in Solea senegalensis larvae by means of histological and immunohistochemical techniques. The first thyroid follicle was present at 4 days-post-hatch (dph) coinciding with first feeding. During metamorphosis (12–20 dph) the follicles increased in both number and size, and by 30 dph presented the same characteristics as that seen in adult fish. Tissue immunostaining of both thyroid hormones decreased during the endogenous larvae development to nearly undetectable levels at the completion of yolk-sac absorption. During larvae exogenous phase, T3 and T4 immunostaining was first detected by 6 dph and an increase of specific staining for both hormones was detected between 12 and 20 dph, during metamorphosis phase.

The growth and survival of the polychaete Nereis diversicolor (O. F. Müller, 1776) fed on faeces of the carpet shell clam Ruditapes decussatus (L., 1758) were studied under controlled temperature and salinity conditions. Juvenile worms were fed with clam R. decussatus faeces in the presence or absence of sediment during 65 days. Starvation treatments were tested as a control. The results of the present study show that the worms were able to grow using the clam faeces in the presence or absence of sediment. However, the worms from the trials with sediment showed a higher survival rate and biomass production than those from the trials in which no sediment was used. These findings suggest that a suspension of bivalve faeces can be used in the rearing of juvenile N. diversicolor in an integrated polyculture system., El crecimiento y la supervivencia del poliqueto Nereis diversicolor (O. F. Müller, 1776) alimentado con heces de almeja Ruditapes decussatus (L., 1758) fueron estimados bajo condiciones controladas de temperatura y salinidad. Los poliquetos juveniles fueron alimentados con heces en presencia y ausencia de sedimento durante 65 días. Como control se aplicaron tratamientos de ayuno. Los resultados sugieren que los poliquetos crecieron usando las heces como única dieta en presencia y en ausencia de sedimento. Es más: los poliquetos de los ensayos con sedimiento mostraron supervivencia y producción de biomasa mayores que aquéllos en que no se utilizó sedimiento. Los resultados sugieren que se puede usar una suspensión de heces procedentes de bivalvos en el cultivo de juveniles de N. diversicolor en un sistema de policultivo integrado.

A 65-day experiment was conducted to determine survival, growth, feed utilization and oogenesis of Nereis diversicolor (O. F. Müller, 1776), fed with a commercial dry diet developed for gilthead seabream Sparus auratus L., 1758. Worms were reared in three replicate tanks with 50 worms per tank (255 indiv · m_2). Tetramin®, a commonly used diet for ornamental fish and polychaete laboratory experiments, was used as a control. Survival was high (95.3 %) for both treatments, and not significantly affected by diet. Specific growth rates and feed efficiency ratios did not differ significantly between worms fed with either diets, nor were there significant differences in the proportion of individuals in the oocyte size classes., La supervivencia, el crecimiento, la utilización del alimento y la oogénesis de Nereis diversicolor (O. F. Müller, 1776) alimentado con pienso de dorada Sparus auratus L., 1758 fueron estudiados durante 65 días. Los poliquetos se cultivaron en tres tanques replicados con 50 individuos en cada uno (255 indiv · m_2). Como control se utilizó Tetramin®, una dieta común para peces ornamentales y en experimentos de laboratorio con poliquetos. La supervivencia fue alta (95,3 %) en ambos tratamientos y no se vio afectada significativamente por la dieta. El aumento de peso y los cocientes de eficiencia alimentaria no variaron significativamente entre los poliquetos alimentados con ambas dietas. Entre los grupos experimentales no hubo diferencia significativa en la proporción de individuos por clases de tamaño de los oocitos.