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Cortisol is the main glucocorticoid hormone promoting compensatory metabolic responses of stress in teleosts. This hormone acts through genomic and membrane-initiated actions to exert its functions inside the cell. Experimental approaches, using exogenous cortisol administration, confirm the role of this hormone during short (minutes to hours)- and long-term (days to weeks) responses to stress. The role of membrane-initiated cortisol signaling during long-term responses has been recently explored. In this study, Sparus aurata were intraperitoneally injected with coconut oil alone or coconut oil containing cortisol, cortisol-BSA, or BSA. After 3 days of treatment, plasma, liver, and skeletal muscle were extracted. Plasma cortisol, as well as metabolic indicators in the plasma and tissues collected, and metabolism-related gene expression, were measured. Our results showed that artificially increased plasma cortisol levels in S. aurata enhanced plasma glucose and triacylglycerols values as well as hepatic substrate energy mobilization. Additionally, cortisol stimulated hepatic carbohydrates metabolism, as seen by the increased expression of metabolism-related genes. All of these responses, observed in cortisol-administered fish, were not detected by replicating the same protocol and instead using cortisol-BSA, which exclusively induces membrane-initiated effects. Therefore, we suggest that after three days of cortisol administration, only genomic actions are involved in the metabolic responses in S. aurata., This work was funded by Spanish Ministry of Science and Innovation—MICINN (PID2020-117557RB-C22) awarded to JMM; Fondo de Financiamiento de Centros de Investigación en Áreas Prioritarias (FONDAP) Grant INCAR 15110027; Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) Grant 1201498 (to Juan Antonio Valdés), and ANID FONDECYT Postdoctorado [grant number 3210050] awarded to Jorge E. Aedo. The authors (I.R, G.M, and J.M.M) belong to the Fish Welfare and Stress Network (AGL2016-81808-REDT), supported by the Agencia Estatal de Investigación (MICINN, Spanish Government).

Evaluating the survival of discarded species is gaining momentum after the new European Common Fisheries Policy (Article 15 of the European Regulation No. 1380/2013). This regulation introduced a discard ban, with an exemption for those species with demonstrated high survival rates after their capture and release. Candidate species should be evaluated for each fishing gear and geographical area. In this study, we assessed not only survival, but also physiological recovery rates of blackspot seabream (Pagellus bogaraveo) below commercial size captured with a hookline called “voracera” in the Strait of Gibraltar (SW Atlantic waters of Europe). Experiments onboard a commercial fishing vessel were paralleled with studies in controlled ground-based facilities, where the capture process was mimicked, and physiological recovery markers were described. Our results confirmed that hookline capture induced acute stress responses in the target species, such as changes in plasma cortisol, lactate, glucose, and osmolality. However, 90.6% of the blackspot seabreams below commercial size captured with this fishing gear managed to survive, and evidenced physiological recovery responses 5 h after capture, with complete homeostatic recovery occurring within the first 24 h. Based on this study, the European Commission approved an exemption from the discard (EU Commission Delegated Regulation 6794/2018). Thus, the robust methodology described herein can be an important tool to mitigate the problem of discards in Europe.

In aquaculture facilities fish welfare could be compromised due to stressors. Fish deal with stress, inter alia, through the activation of the hypothalamic-pituitary-interrenal endocrine axis and, as a result, corticosteroids are released into the blood. Recent studies have described that corticosteroids actions depend on the specific affinities to their receptors, and the subsequent differentiated responses. Cortisol is the main corticosteroid hormone in teleost fish, being its actions dependent on the intensity and time of exposure to stressors. Short-term effects of corticosteroids are well described, but long-term effects, including changes in the energy management directly affecting growth and survival, are less understood in fish. Here we show the effects of chronic oral administration of cortisol and the synthetic glucocorticoid dexamethasone (DXM) on the intermediary metabolism of the gilthead seabream (Sparus aurata). We described a higher energy expenditure associated to both corticosteroids resulting in lower growth rates of fish. Moreover, the effects of these compounds were tissue-dependant, with differences between both hormones. Thus, cortisol-fed animals accumulated triglycerides in the liver, while DXM treatment led to glycogen storage. Cortisol and DXM stimulated amino acids catabolism and gluconeogenic pathways in muscle and gills, but the effects were significantly enhanced in DXM-fed fish. The described effects highlighted differentiated mechanisms of action associated to both corticosteroids under chronic stress conditions. Further studies should aim at describing those pathways in detail, with special attention to the functionality of glucocorticoid receptor isoforms. The effects described here for S. aurata juveniles, may serve as a basis to assess long-term stress in future comparative studies with other aquaculture species.

The use of lysed microalgae in the diet of carnivorous fish can increase the bioavailability of proteins and bioactive compounds, such as unsaturated fatty acids or vitamins in the digestive tract. These are essential molecules for the proper physiological development of fish in aquaculture. However, some antinutritional components and other undesirable molecules can be released from an excess of microalgae supplied, compromising the integrity of the intestine. The inclusion of small amounts of hydrolized microalgae in the fish diet can be a good strategy to avoid negative effects, improving the availability of beneficial compounds. Nannochloropsis gaditana is an interesting microalgae as it contains nutraceuticals. Previous studies reported beneficial effects after its inclusion in the diet of Sparus aurata, a widely cultured species in Europe and in all Mediterranean countries. However, administration of raw microalgae can produce intestinal inflammation, increased intestinal permeability, bacterial translocation and disturbance of digestion and absorption processes. The aim of this study was to evaluate changes in the intestinal microbiota and barrier stability of S. aurata fed with low inclusion (5%) hydrolysed N. gaditana. Intestinal microbiota was analyzed using Illumina MiSeq technology and libraries were constructed using variable regions V3–V4 of 16S rDNA molecules. Analysis were based in the identification, quantification and comparison of sequences. The predictive intestinal microbial functionality was analyzed with PICRUSt software. The results determined that the intestinal microbiota bacterial composition and the predictive intestinal microbiota functionality did not change statistically after the inclusion of N. gaditana on the diet. The study of gene expression showed that genes involved in intestinal permeability and integrity were not altered in fish treated with the experimental diet. The potential functionality and bacterial taxonomic composition of the intestinal microbiota, and the expression of integrity and permeability genes in the intestine of the carnivorous fish S. aurata were not affected by the inclusion of hydrolysed 5% N. gaditana microalgae.

Stress responses in teleosts include the release of hormones into the bloodstream. Their effects depend on the species and on the environmental conditions. The Amazon basin collects waters of diverse chemical composition, and some fish are able to inhabit several of them. However, the effects of these waters on the stress axis are still unknown. Here we show how acute air-exposure differently affects stress biomarkers in tambaqui (Colossoma macropomum), a tropical model species, when acclimated to two Amazonian waters (Rio Negro -RN- water rich in humic acids and poor in ions, and groundwater -IG- with no humic acids and higher concentration of ions). This study described primary and secondary stress responses after air exposure including plasma cortisol, energy metabolites, pH and ions, skin mucus energy metabolites, as well as gills and kidney Na+/K+-ATPase and Na+/H+-exchanger (NHE) activities. Several differences were described in these stress biomarkers due to the acclimation water. The most remarkable ones include increased mucus glucose only in RN-fish, and mucus lactate only in IG-fish after air exposure. Moreover, an inverse relationship between plasma cortisol and Na+ concentrations as well as a direct relationship between plasma ammonia and branchial NHE activity were observed only in RN-fish. Our results demonstrate how important is to study stress responses in fish acclimated to different environments, as physiological differences can be magnified during episodes of high energy expenditure. In addition to having a direct application in aquaculture, this study will improve the management of critical ecosystems such as the Amazon.

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psμ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na+,K+-ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na+,K+-ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (Vmax) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na+,K+-ATPase capacity appears fixed and activity appears to be determined by temperature., This study was partly supported by grants AGL2007-61211/ACU (Ministerio de Innovación y Ciencia and FEDER, Spain) and Proyecto de Excelencia PO7-RNM-02843 (Junta de Andalucía) to JMM. L.V.C. is funded FONDAP-IDEAL 15150003.

In 2019, Europe will adopt a no-discards policy in fisheries. This entails the landing of captured species unless strong evidence is provided supporting their survival and recovery after fishing. Thus, research on this topic is gaining momentum. Bottom-trawling, as a non-selective fishing method, is characterized by a high proportion of discards including vulnerable key species, such as demersal sharks. Their survival may also depend on capture depth. By paralleling onboard and laboratory experiments with the small-spotted catshark, Scyliorhinus canicula, we offer a robust experimental design to assess the survival of discarded sharks. Catsharks were captured by bottom-trawling at two depths (shallow similar to 89 m and deep similar to 479 m). Blood samples were collected following trawl capture and analyzed for stress biomarkers (lactate, osmolality, phosphate, urea). During recovery in onboard tanks, behavior was video-recorded and fish were re-sampled after 24 h. A second experiment was conducted in laboratory facilities to simulate air-exposure after trawling and to analyze the physiological recovery. Our results showed that 95.7% of the animals survived 24 h after trawling. We confirmed that trawling elicited acute stress responses in catshark but that they managed to recover. This was demonstrated by lactate concentrations that were 2.6 mM upon capture, but recovered to assumed baselines after 24 h (0.2 mM). Non-invasive video monitoring revealed behavioral differences with depth, whereby those captured at 89 m depth required longer to recover than those captured at 479 m depth. Implementation of standardized survival studies by fishery managers can benefit from holistic physiological approaches, such as the one proposed here. European UnionEuropean Union (EU) PADI Foundation [28467] Ministerio de Economia y CompetitividadSpanish Government [AGL2013-48835-C2-R, AGL2016-76069-C2-1-R] Project SUREDEPAR (Programa Pleamar, Ministerio de Agricultura y Pesca, Alimentacion y Medio Ambiente) Ministry of Science and Higher EducationMinistry of Science and Higher Education, Poland European Social Funds through the Portuguese National Science Foundation (FCT)Portuguese Foundation for Science and Technology [PTDC/MAR-BIO/3034/2014] FCTPortuguese Foundation for Science and Technology [UID/Multi/04326/2013]

Transport processes between aquaculture facilities activate the stress response in fish. To deal with these situations, the hypothalamic-pituitary-interrenal (HPI) axis releases cortisol, leading to an increase in circulating energy resources to restore homeostasis. However, if the allostatic load generated exceeds fish tolerance limits, stress-related responses will compromise health and welfare of the animals. In this context, anesthetics have arisen as potential agents aiming to reduce negative effects of stress response. Here we assessed the effects of a sedative dose of clove oil (CO) and MS-222 on hallmarks involved in HPI axis regulation and energy management after simulated transport, and further recovery, in gilthead seabream (Sparus aurata L.) juveniles. Fish were placed in a mobile setup of water tanks where transport conditions were simulated for 6 h. Sedation doses of either CO (2.5 mg L−1) or MS-222 (5 mg L−1) were added in the water tanks. A control group without anesthetics was also included in the setup. Half of the animals (n = 12 per group) were sampled immediately after transport, while remaining animals were allowed to recover for 18 h in clean water tanks and then sampled. Our results showed that the HPI axis response was modified at peripheral level, with differences depending on the anesthetic employed. Head kidney gene-expressions related to cortisol production (star and cyp11b1) matched concomitantly with increased plasma cortisol levels immediately after transport in CO-sedated fish, but these levels remained constant in MS-222-sedated fish. Differential changes in the energy management of carbohydrates, lipids and amino acids, depending on the anesthetic employed, were also observed. The use of CO stimulated amino acids catabolism, while MS-222-sedated fish tended to consume liver glycogen and mobilize triglycerides. Further studies, including alternative doses of both anestethics, as well as the assessment of time-course HPI activation and longer recovery periods, are necessary to better understand if the use of clove oil and MS-222 is beneficial for S. aurata under these circumstances., This work was funded by Spanish Ministry of Science, Innovation and Universities - MICINN (AGL2013-48835-C2-1-R and AGL2016-76069-C2-1-R) awarded to JM. IJ-C is currently supported by a contract of Trainee Predoctoral Research Staff from the University of Cádiz (PIF UCA/REC02VIT/2014; 2018-011/PU/AY.PUENTE/CD). The authors (IJ-C, IR-J, JM-S, and JM) belong to the Fish Welfare and Stress Network (AGL2016-81808-REDT), supported by the Agencia Estatal de Investigación (MICINN, Spanish Government).

The improvement of aquaculture industry is directly linked to the improvement of fish welfare, as better growth rates are related to low-stress conditions for fish. In aquaculture facilities, routine processes such as live transportation activate the stress response compromising welfare of fish. As a solution, the use of anesthetics seems to reduce the negative effects of stress on fish physiology. There is a plethora of anesthetic compounds available, but their effects are however species specific. In this study, the effects of sedative concentrations of AQUI-S® and etomidate on physiological stress responses after a mimicked transport process, and further recovery, were evaluated. Gilthead seabream (Sparus aurata) was studied due to its great importance in European aquaculture. Prior to the transport assay, induction times to sedation, anesthesia and recovery were characterized for different concentrations of each anesthetic. Our results showed that AQUI-S® is more effective to induce deep anesthesia and require shorter recovery times than etomidate. Subsequently, seabream juveniles were placed in a mobile setup of water tanks, and transported for 6 h with a sedative concentration of either AQUI-S® (2 ppm of isoeugenol), etomidate (0.15 ppm), or without anesthetic. Afterwards, all groups were allowed to recover for 18 h in clean water tanks. Samples of plasma, liver and gills were collected before and after the transport process, as well as after the recovery period. The sedative concentrations of both anesthetics evoked differential responses in plasma and liver, and seemed to reduce secondary stress responses in gills compared to the control group. AQUI-S® induced additional plasmatic stress responses (increasing cortisol, glucose and osmolality) after transport. However, etomidate resulted more effective in the transport by reducing energy expenditure and promoting lipid synthesis in the liver. In conclusion, AQUI-S® resulted more suitable in short-term procedures where deep anesthesia is required; while etomidate was more effective to minimize stress responses in 6 h-transported seabream juveniles.

the intestine of marine fishes contributes to the ocean carbon cycle producing carbonate aggregates as part of the osmoregulatory process. Therefore, this study aimed to evaluate physiological adjustments of European sea bass (Dicentrarchus labrax) intestine to a higher pCO(2) environment likely in the near future (similar to 1700 mu atm). At the whole-body level, hypercapnia for 5 weeks resulted in fish having a significantly diminished specific growth rate, condition factor and hepatosomatic index. An increase in plasma osmolality and HCO3- concentration was detected, paralleled by decreased metabolites concentrations. In the intestine, high seawater pCO(2) was without effect on ouabain-sensitive ATPase activities, while Bafilomycin A1-sensitive ATPase activity significantly decreased in the anterior intestine. Anterior and mid intestine were mounted in Ussing chambers in order to measure bioelectrical parameters and bicarbonate secretion by pH-Stat ex-vivo. Hypercapnia induced a 2.3 and 2.8-fold increase in bicarbonate secretion rates in the anterior and mid intestine, respectively. In the intestinal fluid, HCO3- concentration increased 2.2-fold, and carbonate precipitates showed a 4.4-fold increase in response to hypercapnia, paralleled by a > 3-fold increase of drinking and a > 2-fold increase of intestinal volume at any given time. At the molecular level, hypercapnia elicited higher intestinal mRNA expression levels for atp6v1b (V-ATPase B subunit), slc4a4, slc26a3, and slc26a6, both in the anterior and mid intestine. As a whole, our results show that the intestine of sea bass responds to high seawater pCO(2), a response that comes at a cost at the whole-body level with an impact in the fish specific growth rate, condition factor, and hepatosomatic index. Ministry of Science and Higher Education, Portugal European Social Funds through the Portuguese National Science Foundation (FCT)[PTDC/MAR-BIO/3034/2014] Portuguese Foundation for Science and Technology (FCT) [UID/Multi/04326/2019] info:eu-repo/semantics/publishedVersion

Fish welfare is gaining momentum due to consumers' trends and legal regulations. Therefore, aquaculture industry is in the way of mitigating stress to the animals and, thus, improving the physiological status, with the consequent increase in economical profits. As a consequence, feed additives have been proposed as potential stress relievers through the diet. However, the effectiveness of these extracts on stress responses and growth regulation is still to be defined for different species and stressors. Here we show the effects of the commercial natural-additive “Relaquax” on growth performance, endocrine regulation of hypothalamic-pituitary-interrenal (HPI) and somatotropic (GH/IGF-I) axes, as well as intermediary metabolism, of gilthead seabream (Sparus aurata L.) reared in low and high stocking density conditions. For this purpose, S. aurata juveniles were maintained for 3 months at different combined conditions of feeding (control diet or supplemented with the additive) and initial stocking density (low or high; 4.0 or 11.5 kg m−3, respectively). At the end of the experimental period, brain, pituitary, head kidney and liver gene expression of endocrine factors involved in stress and growth regulation were determined. In addition, liver and muscle enzyme activities of the intermediary metabolism of carbohydrates, lipids and amino acids were assessed. Our results showed an increased growth performance in fish fed with the additive, mediated through hepatic igf1 enhanced expression at both stocking densities. In the same way, higher body mass was associated to inhibition of lipid catabolism in muscle (decreased GPDH and HADH activities), as a consequence of diet supplementation. The additive has also reduced amino acid catabolism in liver (decreased AST activity), as well as enhanced glycolysis in muscle (increased PK activity). In conclusion, this additive was confirmed as a natural alternative to improve aquaculture and welfare of seabream, especially under high stocking density conditions., This work was funded by Spanish Ministry of Science and Innovation - MICINN (AGL2013-48835-C2-1-R and AGL2016-76069-C2-1-R) awarded to JMM. IJC was supported by a contract from the University of Cádiz (PIF UCA/REC02VIT/2014; 2018-011/PU/AY.PUENTE/CD). The authors (IJC, IR-J, and JMM) belong to the Fish Welfare and Stress Network (AGL2016-81808-REDT), supported by the Agencia Estatal de Investigación (MICINN, Spanish Government).

The European Fisheries Policy aims at a progressive elimination of discards. An exception from this regulation includes the release of species with high survival rates after capture. In south-western Atlantic waters of Europe, Norway lobster (Nephrops norvegicus) and spottail mantis shrimp (Squilla mantis) are amongst the most important crustacean species captured by bottom trawling. We evaluated their short-term survival probability, survival rates and recovery capacities after being trawled by an oceanographic vessel. Seasonal differences were considered by sampling in spring and autumn. In order to characterise the full recovery after capture, physiological responses were also analysed along a time-course of 24 h. Our results confirm that bottom trawling is a stressful process to these crustacean species, as seen by changes in plasma and muscle metabolites, hemocyanin and immune system parameters. However, maintaining captured animals in onboard water tanks evidenced the full physiological recovery of survivors after 6 h and before 24 h. Survival in Norway lobster and spottail mantis shrimp varied according to the season, being higher in spring (68.4 � 7.1% and 87.0 � 4.7%, respectively) than in autumn (33.8 � 7.8% and 63.8 � 9.3%, respectively), probably due to the higher temperatures registered after summer months. The employment of the presented techniques for the evaluation of other crustaceans, fishing gears and geographical areas can be contemplated. Fisheries stakeholders might use this approach to better manage discards in Europe, Sí

Eleginops maclovinus is a native species with potential for Chilean aquaculture. However, its optimal stocking density and the impact on its physiology is unknown. The aim of this study is to determine the optimal stocking density range by evaluating parameters of growth, stress, intermediary metabolism, and osmoregulation. For this, 48 immature male fish (201.60 ± 32 g; 29.05 ± 1.45 cm) were randomly assigned to four stocking densities (3, 6, 12 and 24 kg/m3) and maintained for 60 days. No mortality was recorded, optimal growth was observed at a density of 3–6 kg/m3 and decreased with a higher stocking density. In plasma, glucose and protein levels were not affected by stocking density, while triglycerides, lactate, total amino acids, cortisol, and osmolality showed a significant decrease at a higher stocking density. Enzymatic activity of branchial and renal NKA, in addition to water content in muscle, increased at a greater stocking density, while intestinal NKA showed non-significant differences. The metabolism of carbohydrates, triglycerides, and amino acids/protein in liver, gill, kidney, and muscle was significantly affected by stocking density and showed significant tissue-specific responses. Glycogen levels increased at a higher stocking density in gills, kidney, and muscle, suggesting that glucose plays an important role against chronic stress produced by stocking density. The GH/IGF-1 axis and HSP70 showed significantly lower levels of mRNA transcript at the high density of 24 kg/m3. In conclusion, we suggest that stocking density affects the physiology of Eleginops maclovinus, activating the stress system and mobilizing energy substrates in a tissue-specific manner. Additionally, our data suggests that male E. maclovinus should be cultivated in the range of 3–12 kg/m3, avoiding the high density of 24 kg/m3.

Land use carries implications for freshwater fish conservation. Plantation forestry practices have been shown to have negative impacts on resident fish fauna, but little work has been conducted to assess these impacts on invasive vs. native fish populations. Ten headwater catchments in the Mediterranean climate zone of Chile were used to assess the impacts of land use (pine plantations vs. native forests) on fish condition (length-weight relationship) and abundance (catch-per-unit-effort (CPUE)) of the invasive trout Oncorhynchus mykiss and the threatened native catfish Nematogenys inermis. Negative impacts on trout condition were associated with a lack of canopy cover and river topology. The presence of N. inermis was associated with catchment factors less favourable to trout. Current environmental regulations and forestry management practices do not appear to create negative pressures on invasive trout from land use practices, despite expectations from the literature. Assessing how land use management regulations impact invasive and native fishes should be a part of species conservation and territorial planning.

In this study we assessed the influence of three different environmental salinities (5, 15 and 31 psu during 90 days) on growth, osmoregulation, energy metabolism and digestive capacity in juveniles of the Notothenioid fish Eleginops maclovinus. At the end of experimental time samples of plasma, liver, gill, intestine, kidney, skeletal muscle, stomach and pyloric caeca were obtained. Growth, weight gain, hepatosomatic index and specific growth rate increased at 15 and 31 psu and were lower at 5 psu salinity. Gill Na(+), K(+)-ATPase (NKA) activity presented a "U-shaped" relationship respect to salinity, with its minimum rates at 15 psu, while this activity correlated negatively with salinity at both anterior and posterior intestinal portions. No significant changes in NKA activity were observed in kidney or mid intestine. Large changes in plasma, metabolite levels and enzymatic activities related to energy metabolism in liver, gill, intestine, kidney and muscle were generally found in the groups exposed to 5 and 31 psu compared to the 15 psu group. Only the pepsin activity (digestive enzymes) assessed enhanced with environmental salinity, while pyloric caeca trypsin/chymotrypsin ratio decreased. This study suggests that juvenile of E. maclovinus presents greater growth near its iso-osmotic point (15 psu) and hyperosmotic environment (31 psu). Acclimation to low salinity increased the osmoregulatory expenditure as seen by the gill and anterior intestine results, while at high salinity, branchial osmoregulatory activity was also enhanced. This requires the mobilization of lipid stores and amino acids, thereby holding the growth of fish back. The subsequent reallocation of energy sources was not sufficient to maintain the growth rate of fish exposed to 5 psu. Thus, E. maclovinus juveniles present better growth efficiencies in salinities above the iso-osmotic point and hyperosmotic environment of this species, showing their best performance at 15 psu as seen by the main osmoregulatory and energy metabolism enzymatic activities.

The aim of this study was to evaluate the inclusion of two macroalgae as dietary ingredients on growth, body composition, intermediate metabolism, intestinal enzymes and gut histology of Sparus aurata juveniles. Six isocaloric and isonitrogenous diets incorporating 5%, 15% or 25% of Gracilaria cornea or Ulva rigida meal were tested against a control diet. Duplicate groups of fifteen fish (14 g) were fed the experimental diets for 70 days. Growth performance showed an inverse linear relationship with the inclusion level of Gracilaria. Feed conversion ratio increased significantly in fish fed 25% Gracilaria. Ulva presented a quadratic relationship with the lowest SGR at 15% of inclusion. Fish fed 25% Ulva exhibited significantly lower lipid content than those fed control diet. Overall, metabolites did not show differences with specimens fed control diet, except for plasmatic glucose and muscle lactate. Intestinal proteolytic activity was reduced with the macroalgae inclusion level, but all fish showed the same enzyme pattern. The histological study revealed no negative effect on liver and intestine structure by the macroalgae inclusion. The present work demonstrated that these ingredients can be used in practical diets for sea bream, but a more comprehensive research is needed to determine the optimum dietary level in a longer feeding trial.

The influence of stocking density and food deprivation on energy metabolism, stress processes and the pituitary endocrine system of silver catfish (Rhamdia quelen) was investigated after a period of 14 days, in which plasmatic and hepatic parameters and the mRNA expression of prolactin (PRL), growth hormone (GH) and somatolactin (SL) were assessed. The fish were subjected to four experimental conditions: (1) fed under high stocking density (32 kg/m3, HSD); (2) fed under mean stocking density (16 kg/m3, MSD); (3) fed under low stocking density (8 kg/m3, LSD); and (4) food-deprived under low stocking density (8 kg/m3, LSD-FD). After 14 days, plasma and liver samples were obtained to analyze the metabolite levels and enzymatic activities related to metabolism, and pituitary glands were obtained to analyze hormone expression (PRL, GH and SL). Liver weight and the hepato-somatic index (HSI) revealed that specimens maintained at HSD and/or MSD had higher hepatic stores, which were observed in the triglyceride and glycogen levels in this tissue, than animals submitted to the LSD and LSD-FD groups. Triglyceride levels in the plasma and liver revealed the consumption of fatty acid reserves in the fasting group. Enzymatic activities, such as glutamate dehydrogenase (GDH), phosphorylase (GPase), pyruvate kinase (PK), aspartate transaminase (AST) and glycerol-3-phosphate dehydrogenase (G3PDH), indicated an increase in gluconeogenic pathways in the HSD group and an increase in glycolitic metabolism in the LSD groups. The expression of PRL was not affected by stocking density and/or food deprivation and GH decreased with increased density and increased in fasting conditions. A negative effect of density and fasting was observed on the expression of SL. Overall, the data suggested that juvenile silver catfish reared at stocking densities of 16 to 32 kg/m3 were better maintained than those maintained at the lowest density., We would like to thank the financial support by CAPES (Ministry of Education of Brazil, Brazil) through the program PDSE-CAPES (Programa Institutional de Bolsas de Doutorado Sanduíche no Exterior — Process: 1585/12-6) to C.M., the CNPq research fellowships to V.L.L. and B.B., the project AGL2010-14876 (Ministry of Science and Education, Spain) to J.M.M. and the program FPU (Formación de Profesorado Universitario — Ref. AP2008-01194) (Ministry of Science and Education, Spain) to JAM-S.

Osmoregulation in fish is a complex process that requires the orchestrated cooperation of many tissues. In fish facing hyperosmotic environments, the intestinal absorption of some monovalent ions and the secretion of bicarbonate are key processes to favor water absorption. In the present study, we showed that bicarbonate levels in the intestinal fluid are several fold higher in seawater than in freshwater acclimated tilapia (Oreochromis mossambicus). In addition, we analyzed gene expression of the main molecular mechanisms involved in HCO3- movements i.e. slc26a6, slc26a3, slc4a4 and v-type H-ATPase sub C in the intestine of tilapia acclimated to both seawater and freshwater. Our results show an anterior/posterior functional regionalization of the intestine in tilapia in terms of expression patterns, which is affected by environmental salinity mostly in the anterior and mid intestine. Analysis of bicarbonate secretion using pH-Stat in tissues mounted in Ussing chambers reveals high rates of bicarbonate secretion in tilapia acclimated to seawater from anterior intestine to rectum ranging between similar to 900 and similar to 1700 nmol HCO3- cm(-2) h(-1). However, a relationship between the expression of slc26a6, slc26a3, slc4a4 and the rate of bicarbonate secretion seems to be compromised in the rectum. In this region, the low expression of the bicarbonate transporters could not explain the high bicarbonate secretion rates here described. However, we postulate that the elevated v-type H-ATPase mRNA expression in the rectum could be involved in this process. (C) 2017 Elsevier Inc. All rights reserved. University of Cadiz, Spain FCT, Portugal [SFRH/BD/113363/2015] Portuguese Foundation for Science and Technology (Ministry of Science and Higher Education, Portugal) [PTDC/MAR-BIO/3034/2014] Portuguese Foundation for Science and Technology (European Social Funds) [PTDC/MAR-BIO/3034/2014] national funds from the Portuguese Foundation for Science and Technology (FCT) [UID/Multi/04326/2013]

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., This study was funded by project AGL2010-14876 from Ministerio de Ciencia e Innovación awarded to J. M. M. (Spain). This work was partially supported by Ph.D. scholarship from the University of Cadiz (UCA 2009-074-FPI) awarded to I. R-J.

Marine fish contribute to the carbon cycle by producing mineralized intestinal aggregates generated as by-products of their osmoregulation. Here we aimed at characterizing the control of intestinal aggregate production in the gilthead sea bream in response to near future increases of environmental CO2. Our results demonstrate that hypercapnia (800 and 1200 μatm CO2) elicits higher intestine epithelial HCO3−secretion and the subsequent parallel increase of intestinal aggregate production when compared to present values (400 μatm CO2). Intestinal gene expression analysis revealed the up-regulation of crucial transport mechanisms involved not only in the intestinal secretion cascade (Slc4a4, Slc26a3 and Slc26a6) of sea bream, but also in other mechanisms involved in intestinal ion uptake linked to water absorption such asNKCC2and theAquaporin 1b.These results highlight the important role of fish in the marine carbon cycle, and their potential growing impact of intestinal biomineralization processes in the scenario of ocean acidification.

Fish are continuously forced to actively absorb or expel water and ions through epithelia. Most studies have focused on the gill due to its role in Na+ and Cl- trafficking. However, comparatively few studies have focused on the changing function of the intestine in response to external salinity. Therefore, the present study investigated the main intestinal changes of long-term acclimation of the Senegalese sole (Solea senegalensis) to 5,15, 38 and 55 ppt. Through the measurement of short-circuit current (Isc) in Ussing chambers and biochemical approaches, we described a clear anterior/posterior functional regionalization of the intestine in response to salinity. The use of specific inhibitors in Ussing chamber experiments, revealed that the bumetanide-sensitive Na+/K+/Cl- co-transporters are the main effectors of Cl- uptake in both anterior intestine and rectum. Additionally, the use of the anion exchanger specific inhibitor, DIDS, showed a salinity/region dependency of anion exchanger function. Moreover, we also described ouabain-sensitive Na+/K+-ATPase (NKA) and Bafilomycin Al-sensitive H-ATPase activities (HA), which displayed changes related to salinity and intestinal region. However, the most striking result of the present study is the description of an omeprazole-sensitive H+/K+-ATPase (HKA) in the rectum of Senegalese sole. Its activity was consistently measurable and increased at lower salinities, reaching rates even higher than those of the NKA. Together our results provide new insights into the changing role of the intestine in response to external salinity in teleost fish. The rectal activity of HKA offers an alternative/cooperative mechanism with the HA in the final processing of intestinal water absorption by apical titration of secreted bicarbonate. (C) 2016 Elsevier Inc. All rights reserved. Ministry of Science and Education, Spain [AGL2010-14876] Ministry of Science and Higher Education European Social Funds through the Portuguese National Science Foundation (FCT) [PTDC/MAR-BIO/3811/2012, PTDC/MAR-BIO/3034/2014] Portuguese Foundation for Science and Technology (FCT) [UID/Multi/04326/2013]

The aim of this study was to evaluate immunological, metabolic and osmoregulatory secondary stress responses in Eleginops maclovinus specimens submitted to three different stocking densities: i) low (3.1 kg m(-3)), medium (15 kg m(-3)) and high (60 kg m(-3)) during 10 days, alone or in combination with a previous treatment of a protein extract of the pathogen Piscirickettsia salmonis (0.5 μg g weight body(-1)). Plasma, liver, gill and kidney samples were obtained at the end of both experiments. Plasma cortisol and amino acid levels increased, while plasma glucose, triglyceride and lactate levels decreased at higher stocking densities. However, no effects were observed on serum Immunoglobulin type M (IgM anti P. salmonis level) values. Gill Na(+), K(+)-ATPase activity enhanced under these experimental conditions, suggesting an osmotic imbalance. Energy metabolism changes, assessed by metabolite concentrations and enzyme activities, indicated a reallocation of energetic substrates at higher stocking densities. Specimens inoculated with a protein extract of P. salmonis and maintained at different stocking densities showed primary stress response, as all groups enhanced plasma cortisol concentrations. Serum IgM levels increased after treatment with P. salmonis extract but a negative influence of high stocking density on IgM production was observed when immune system was activated. Furthermore, treatment with P. salmonis protein extract evoked deep changes in the metabolite stores in all tissues tested, indicating a mobilization of energy substrates in response to infection. The results show that stocking density induced immunological, metabolic and osmoregulatory secondary stress responses in E. maclovinus specimens and that previous treatment with P. salmonis compromise these changes.

In this work, we have assessed the effects of different stocking densities on the biometry, survival and physiological status of the wedge sole (Dicologoglossa cuneata), focusing on changes in the stress system and intermediate metabolism, with the aim of determining a stress indicator for chronic-stress situations in this species. Wedge sole were kept at three different stocking densities (0.2, 0.4 and 0.8 kg m−3) for 197 days, and survival, growth, plasma metabolites, cortisol and enzymatic activities were assessed. Survival rates were the highest at low density, though growth did not vary significantly among treatments. Enzymatic activities, mainly in muscle, differed depending on stocking density. Liver hexokinase activity at low stocking density was the highest, while no differences were detected for the other enzymes assessed. In muscle, all enzymes significantly increased in activity with stocking density. We concluded that long-term high stocking density culture significantly changed enzyme activities (hexokinase, glutamate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase) in wedge sole muscle, although liver enzymes, plasma cortisol and metabolites did not vary significantly. Therefore, muscle enzymes, hexokinase and glutamate dehydrogenase, could be considered stress indicators for this species in chronic-stress situations.

The aim of this study was to carry out a comparative analysis of the osmoregulatory properties and associated energy metabolism of euryhaline flatfish species that are cultured in the world. Culture of flatfish (pleuronectiformes) requires stage- and species-dependent osmotic conditions for rearing. Additionally, geographic origin of broodstock animals is another factor to be taken into account for the culture of pleuronectiformes. Larval and juvenile stages of many flatfish species are cultured in large nurseries situated in estuaries and shallow marine habitats, where the environmental salinity is close to the iso-osmotic point of their internal milieu. This fact implicates an advantage in terms of energy savings for osmoregulatory purposes. Thus, this ‘saved’ energy can be derived to other physiological processes, such as somatic growth. However, this scientific presumption does not always results in an optimal growth for many flatfish species. Indeed, iso-osmotic culture conditions can evoke a higher allostatic load than that in the usual hyper-osmotic environment where flatfish species live wildly. Optimization of flatfish culture thus requires adjustments of the osmotic culture conditions to the specific osmoregulatory and metabolic demands that eventually determine the allostatic load and consequently condition growth rates. In this sense, the geographical location of aquaculture farms and the osmoregulatory-based selection of the species to be cultivated in a particular area become critical factors to be considered for optimal flatfish culture.

Fish aquaculture generates stressful situations that reduce animal welfare conditions and the economic benefits. In this article, we expose two possibilities for stress attenuation in the gilthead seabream ( Sparus aurata L.), an emblematic species for the national and European aquaculture.

Eleginops maclovinus is an endemic, subantarctic Notothenioidei species. This study examined the influence of different environmental salinities (5, 15, and 45 psu; and 32 psu as a control) on energy metabolism in E. maclovinus over a period of 14 days. Metabolite contents and enzymatic activities related to carbohydrate, amino acid, and lipid metabolisms were evaluated in metabolic (liver) and osmoregulatory (gill and kidney) tissues. At extreme salinities (5 and 45 psu), the liver showed a high consumption of energy reserves, mainly as amino acids and carbohydrates. Carbohydrate metabolism in the gills did not change under different salinities, but increased lactate levels were found, suggesting that this tissue may use lactate as an energy substrate. Amino acid metabolism in the gills decreased at 5 psu but increased at 45 psu, and lipid metabolism increased at 5 and 15 psu during the first days of the trial, indicating a possible use of lipids as energy. Kidney carbohydrate catabolism and amino acid metabolism increased after 14 days at 45 psu, while lipid metabolism did not vary in relation to salinity changes. Together, these results suggest that the liver is most affected by salinity changes, probably due to its role as a supplier of energetic substrates. The gills and kidney, osmoregulatory tissues, maintained their energy metabolism levels with minor modifications. In conclusion, E. maclovinus exhibits metabolic adjustments to adapt to different salinities, showing the best responses in isosmotic environmental salinities.

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 55 ppt) for 14 days, 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 (5 ppt) elicited a significant increase in fT3 (29%) and fT4 (184%) plasma concentrations compared to control animals (acclimated to 40 ppt, 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 15 ppt). The high salinity (55 ppt) 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 (40 ppt). 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., This work was partially supported by a Socrates/Erasmus Grant from the European Union and a Ph.D. scholarship from the University of Cadiz (UCA 2009-074-FPI) to I. R-J. It has been also supported by grants AGL2007-61211/ACU (Ministerio de Educación y Ciencia and FEDER, Spain) and Proyecto de Excelencia PO7-RNM-02843 (Junta de Andalucía) to J.M.M. BL (SFRH/BPD/89889/2012) and PISP (SFRH/BPD/84033/2012) were supported by the Science Foundation (FCT) of Portugal.

The influence of culture density on growth, stress system and metabolism has been assessed in A. regius juveniles submitted to different initial stocking densities (3, 7, 10 and 13 g L− 1) for 40 days. On days 0, 12, 27 and 40 of the experiment, biometric parameters were measured. In addition, plasma, liver and muscle samples were taken for biochemical analysis at the end of experimental time. Our results showed better growth rates and feed utilization in those specimens held at high stocking densities when compared to those under low stocking densities. In addition, higher plasma cortisol, glucose and triglycerides levels found in the lowest stocking density tested suggest an activation of the stress system in fish held at this condition. Moreover, no changes in hepatic metabolites were observed, while values of muscle triglycerides, amino acids and lactate enhanced when stocking density increased. In conclusion, the culture of juvenile A. regius at higher initial densities (at least 13 g L− 1) ensures better growth and the optimization of the existing facilities, improving welfare of this species in aquaculture systems., Study funded by project AGL2013-48835-C2-1-R (Ministerio de Economía y Competitividad, Spain) awarded to J.M.M.