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Numerous studies have reported the disadvantages of using plant protein in aquafeeds owing to the presence of antinutritional factors. Particularly, phytate can be overcome by dietary supplementation with exogenous phytase. On the other hand, the use of microalgae has been proven to be a valuable strategy for enhancing the digestive functionality in fish fed diets with high plant protein content. It was hypothesized that the simultaneous inclusion of phytase with microalgae can be useful to improve growth performance and digestive functionality in fish fed diets with high content of plant protein. In this study, the effects of feeds supplemented with different levels of exogenous phytase (FTU) and 2.5% of a blend of Arthrospira platensis and Nannochloropsis gaditana were evaluated on growth performance, metabolism, and gut functionality in European seabass (Dicentrarchus labrax) juveniles. Fish were fed ad libitum for 83 days with five diets containing different enzyme ratios (FTU), in triplicate: (i) control (0 FTU); (ii) 500 FTU; (iii) 1,000 FTU; (iv) 2,000 FTU; and (v) 10,000 FTU. At the end of the feeding period, zootechnical indexes were estimated, and biological samples were withdrawn for physiological determinations. The results obtained showed an overall improvement in the productive efficiency, general metabolism, and intestinal functionality in fish fed the highest phytase inclusion (2,000 and 10,000 FTU). Final weight (75.51 ± 1.01 g vs. 66.76 ± 1.26 g, p = 0.005), SGR (1.15 ± 0.02 vs. 1.00 ± 0.02, p = 0.006). and feed efficiency (0.80 ± 0.02% day−1 vs. 0.67 ± 0.01% day−1, p = 0.012) were higher in fish fed 10,000 FTU. The chemical composition of muscle and plasma metabolites did not vary among dietary treatments, except for the high glucose and low glycogen content in the liver with 2,000 FTU group. Focusing on gut functionality, enzyme activities tended to be higher in fish fed diets supplemented with phytase and microalgae, and significant differences were found for trypsin, chymotrypsin, and alkaline phosphatase with 1,000, 2,000, and 10,000 FTU, and leucine aminopeptidase with 2,000 and 10,000 FTU (p < 0.0001). Analysis of intestinal morphology revealed that 2,000 and 10,000 FTU combined with microalgae increased villi height and decreased villi diameter and enterocyte height. The presence of microalgae at low phytase supplementation level seems to lack of influence on the different parameters evaluated whereas only increased dietary phytase levels impacted on growth, nutrient utilization, and intestinal functionality. In addition, faecal samples were obtained by stripping the final portion of the intestines, from which the phosphorus and nitrogen were quantified. Apparent digestibility coefficient of phosphorus with 2,000 FTU and 10,000 FTU were higher, 74.3% and 77.8%, respectively, compared to control (52.4%). The results revealed a decrease in the amount of P shed with the excreta from the animals supplemented with phytase, a fact that evidenced an increased intestinal absorption of this element. Overall, the results obtained pointed out that phytase can be used at 10,000, and even 2,000, FTU in combination with microalgae as an approach to reducing feed costs and boost fish growth and digestive functionality of fish, while minimizing the environmental impacts of juvenile European seabass farms. [ABSTRACT FROM AUTHOR]

Abstract: The effects of diet supplemented with Myrcia sylvatica essential oil (EOMS) on growth and stress tolerance to different stocking densities were assessed in Sparus aurata. Initially, three experimental groups were established: (i) control, (ii) 1.0 and (iii) 2.0 ml EOMS/kg dry feed. After 90 days, nine fish per group were anaesthetized, weighed and sampled for metabolic analysis. The remaining fish were divided into two different conditions: low (LD—5 kg/m3) and high (HD—40 kg/m3) stocking densities, constituting a final experimental setup with six treatments. After 22 days, 10 individuals per treatment were anaesthetized and sampled for biochemical analysis. EOMS feeding for 90 days did not interfere with growth, although it was effective in reducing cortisol levels and the activation of lipid and carbohydrate metabolism. The HD interfered in growth performance, increased the lactate levels in the control group, reducing also hepatic glycogen levels without dependence on diet supplementation. The 2.0 ml EOMS + LD group increased the growth parameters and decreased the cortisol and triglycerides levels. Additionally, metabolism adjustment was observed to provide energy during LD and HD conditions, indicating that diets supplemented with EOMS produce metabolic reorganization to maintain body homeostasis, suggesting its use as a beneficial supplement in fish. [ABSTRACT FROM AUTHOR]

In euryhaline teleosts, the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-interrenal axes (HPT and HPI, respectively) are regulated in response to environmental stimuli such as salinity changes. However, the molecular players participating in this physiological process in the gilthead seabream (Sparus aurata), a species of high value for aquaculture, are still not identified and/or fully characterized in terms of gene expression regulation. In this sense, this study identifies and isolates the thyrotropin-releasing hormone (trh) mRNA sequence from S. aurata, encoding prepro-Trh, the putative factor initiating the HPT cascade. In addition, the regulation of trh expression and of key brain genes in the HPI axis, i.e., corticotrophin-releasing hormone (crh) and corticotrophin-releasing hormone-binding protein (crhbp), was studied when the osmoregulatory status of S. aurata was challenged by exposure to different salinities. The deduced amino acid structure of trh showed 65-81% identity with its teleostean orthologs. Analysis of the tissue distribution of gene expression showed that trh mRNA is, though ubiquitously expressed, mainly found in brain. Subsequently, regulation of gene expression of trh, crh, and crhbp was characterized in fish acclimated to 5-, 15-, 40-, and 55-ppt salinities. In this regard, the brain gene expression pattern of trh mRNA was similar to that found for the crh gene, showing an upregulation of gene expression in seabream acclimated to the highest salinity tested. Conversely, crhbp did not change in any of the groups tested. Our results suggest that Trh and Crh play an important role in the acclimation of S. aurata to hypersaline environments. [ABSTRACT FROM AUTHOR]

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture. [ABSTRACT FROM AUTHOR]

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture.

Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55ppt) for 14days, and plasma free thyroid hormones (fT3, fT4), outer ring deiodination and Na + /K + -ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5ppt) elicited a significant increase in fT3 (29%) and fT4 (184%) plasma concentrations compared to control animals (acclimated to 40ppt, natural salinity conditions in the Bay of Cádiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit β (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15ppt). The high salinity (55ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (dio1 and dio2) and activity did not change and were similar to controls (40ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The effects of starvation and re-feeding on metabolites and tissue composition, GH/IGF-I axis, and digestive enzyme activities in juvenile thick-lipped grey mullet (Chelon labrosus) were evaluated. Fish were divided into three feeding groups (n=72, 82.00±4.09 g initial body mass). The control group was fed 1% of their body mass once a day throughout the experiment with commercial pellets. The other two groups were deprived of feed for 21 days (starved), or re-fed for 7 days after 14 days of food deprivation (re-fed). Full-length cDNAs from pituitary GH and hepatic IGF-I were cloned by screening a cDNA library or by PCR techniques. Furthermore, changes in their mRNA expressions were assessed by real time PCR in specimens maintained under the different feeding patterns. Results showed a negative growth in starved and re-feeding groups. Starvation induced a significant increase in plasma triglycerides as well as a decrease in liver glucose and glycogen. Re-feeding increased plasma glucose, lactate and protein, as well as liver glucose and glycogen. In addition, starvation significantly increased pituitary GH expression, while re-feeding down-regulated it. No significant changes were observed in hepatic IGF-I expression in any dietary treatment. Digestive enzyme activities were not significantly affected either by starvation or by re-feeding. The results of the present work suggest that juveniles of the thick-lipped grey mullet may easily adjust their metabolism under situations characterized by a short-term starvation and re-feeding., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The dynamic changes in mRNA expression levels for vasotocin (AVT) and isotocin (IT) receptor gene levels were assessed in a time-course response study in immature male specimens of the gilthead sea bream (Sparus aurata) submitted to hyper- (55‰ salinity) and hypo-osmotic (5‰ salinity) challenges. Two different cDNAs for the AVT receptor and one for the IT receptor (V1a2-type and V2-type AVTR, and ITR, respectively) were cloned by screening an S. aurata brain cDNA library. Genes for these receptors were expressed differentially and is nearly ubiquitously in 26 of the examined tissues. In the gills, both environmental salinity challenges up-regulated AVTR V1a2-type gene expression concomitantly with mRNA expression protein activity of Na(+), K(+)-ATPase gene expression and protein, whereas the AVTR V2-type and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels were associated with mRNAs environmental salinity, indicating a possible connection between AVTRs and these transporters. In kidney, AVTR V1a2-type gene expression peaked rapidly and lasted only a short time (12-24h) in response to both osmotic challenges. In contrast, AVTR V2-type mRNA levels were enhanced in specimens exposed to hyperosmotic conditions, whereas they decreased under hypoosmotic environments, suggesting an antidiuretic role related to the vasoconstriction function. In the hypothalamus, only the expression of the AVTR V2-type gene was enhanced at 7 and 14 days under both experimental conditions. In the liver, both AVTRs had increased mRNA levels, with the upregulation of their AVTR V2-type gene increasing faster than the V1a2-type. The ITR gene was not sensitive to variations of external salinity in any of the analyzed tissues. Our results demonstrate the involvement of the vasotocinergic, but not the isotocinergic, pathway as well as the hypothalamic function, in the adjustments of both osmoregulatory and metabolic processes after osmotic challenges., (Copyright © 2013 Elsevier Inc. All rights reserved.)

To investigate the physiological roles of arginine vasotocin (AVT) and isotocin (IT) in osmoregulatory process in gilthead sea bream (Sparus aurata), a time course study (0, 12h, and 1, 3, 7 and 14 days) has been performed in specimens submitted to hypoosmotic (from 40‰ salinity to 5‰ salinity) or hyperosmotic (from 40‰ salinity to 55‰ salinity) challenges. Plasma and liver osmoregulatory and metabolic parameters, as well as AVT and IT pituitary contents were determined concomitantly with hypothalamic pro-vasotocin (pro-VT) and pro-isotocin (pro-IT) mRNA expression levels. Previously, sequences coding for pro-VT and pro-IT cDNAs were cloned. Two osmoregulatory periods related to plasma osmolality and metabolic parameter variations could be distinguished: i) an adaptative period, from 12h to 3 days after transfer, and ii) a chronic regulatory period, starting at day 3 after transfer. Higher values in hypothalamic pro-VT and pro-IT mRNA expression as well as in pituitary AVT and IT storage levels in both hypo- and/or hyper-osmotic transfers have been distinguished. These increase correlated with changes in plasma cortisol levels, suggesting an interaction between this hormone and pro-VT expression. Furthermore, pro-IT expression enhancement also suggests a role of the isotocinergic system as a modulator in the acute stress response induced by hyper-osmotic challenge in S. aurata., (© 2013.)

Facilitative glucose transporters (GLUT) are transmembrane transporter proteins involved in glucose transport across the plasma membrane. In fish, several GLUT mRNAs have been cloned, but to date there is no information about these transporters in the marine euryhaline teleost Sparus aurata. In the present study we obtained the complete coding sequence from S. aurata GLUT1 (saGLUT1), composed by 4483 bases, presenting a 79 to 95% identity with respect to other fish GLUT1 mRNAs. The analysis of the 5' and 3' UTRs showed the presence of several post-transcriptional regulatory elements. In addition, the effect on saGLUT1 mRNA expression of the osmotic acclimation to four different environmental salinities (5, 12, 40 and 55 ppt), in gills, kidney, liver and brain, was studied. Changes in mRNA expression levels were detected in gills and brain, indicating that GLUT1 has an important role in these organs for osmotic acclimation in S. aurata., (Copyright © 2012 Elsevier Inc. All rights reserved.)