Microbiota composition and intestinal integrity remain unaltered after the inclusion of hydrolysed Nannochloropsis gaditana in Sparus aurata diet.

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.

Ocean acidification compromises energy management in Sparus aurata (Pisces: Teleostei).

The effects of ocean acidification mediated by an increase in water pCO2 levels on marine organisms are currently under debate. Elevated CO2 concentrations in the seawater induce several physiological responses in teleost fish, including acid-base imbalances and osmoregulatory changes. However, the consequences of CO2 levels enhancement on energy metabolism are mostly unknown. Here we show that 5 weeks of exposure to hypercapnia (950 and 1800 µatm CO2) altered intermediary metabolism of gilthead seabream (Sparus aurata) compared to fish acclimated to current ocean values (440 µatm CO2). We found that seabream compromises its physiological acid-base balance with increasing water CO2 levels and the subsequent acidification. Intestinal regions (anterior, mid, and rectum) engaged in maintaining this balance are thus altered, as seen for Na+/K+-ATPase and the vacuolar-type H+-ATPase activities. Moreover, liver and muscle counteracted these effects by increasing catabolic routes e.g., glycogenolysis, glycolysis, amino acid turnover, and lipid catabolism, and plasma energy metabolites were altered. Our results demonstrate how a relatively short period of 5 weeks of water hypercapnia is likely to disrupt the acid-base balance, osmoregulatory capacity and intermediary metabolism in S. aurata. However, long-term studies are necessary to fully understand the consequences of ocean acidification on growth and other energy-demanding activities, such as reproduction.

Tambaqui (Colossoma macropomum) acclimated to different tropical waters from the Amazon basin shows specific acute-stress responses.

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.

Survival rates and physiological recovery responses in the lesser-spotted catshark (Scyliorhinus canicula) after bottom-trawling.

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 ~89 m and deep ~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.

What can we learn from glucocorticoid administration in fish? Effects of cortisol and dexamethasone on intermediary metabolism of gilthead seabream (Sparus aurata L.).

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.

High rates of intestinal bicarbonate secretion in seawater tilapia (Oreochromis mossambicus).

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 ~900 and ~1700nmolHCO3-cm-2h-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.

Characterization of the peripheral thyroid system of gilthead seabream acclimated to different ambient 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 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 ß (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.

Intestinal response to salinity challenge in the Senegalese sole (Solea senegalensis).

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 55ppt. 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 A1-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.

Effects on the metabolism, growth, digestive capacity and osmoregulation of juvenile of Sub-Antarctic Notothenioid fish Eleginops maclovinus acclimated at different salinities.

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.

Starving/re-feeding processes induce metabolic modifications in thick-lipped grey mullet (Chelon labrosus, Risso 1827).

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.

Combined effects of high stocking density and Piscirickettsia salmonis treatment on the immune system, metabolism and osmoregulatory responses of the Sub-Antarctic Notothenioid fish Eleginops maclovinus.

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.

Metabolic and osmoregulatory changes and cell proliferation in gilthead sea bream (Sparus aurata) exposed to cadmium.

The impact of cadmium on metabolism and osmoregulation was assessed in gilthead sea bream (Sparus aurata). Seawater acclimated fish were injected intraperitoneally with a sublethal dose of cadmium (1.25 mg Cd/kg body wt). After 7 days, half of the injected fish were sampled. The remaining fish were transferred to hypersaline water and sampled 4 days later. Gill and kidney Na(+)/K(+)-ATPase activities, plasma levels of cortisol, several metabolites and osmolytes, as well as osmolality were measured. Hepatosomatic index and condition factor were calculated. The expression levels of Na(+)/K(+)-ATPase, heat shock proteins (HSP70, HSP90) and proliferating cell nuclear antigen was assessed by western blotting. Cadmium treatment adversely affected the Na(+)/K(+)-ATPase activity, although, there was no perturbation in ion homeostasis and the animals were not compromised following transfer to hypersaline water. Increased cell proliferation and Hsp90 expression likely contributed to the attenuation of the deleterious effects of cadmium exposure.