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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 (V max ) 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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Marine species have colonized extreme environments during evolution such as freshwater habitats. The amphidromous teleost fish, Galaxias maculatus is found mainly migrating between estuaries and rivers, but some landlocked populations have been described in lakes formed during the last deglaciation process in the Andes. In the present study we use mtDNA sequences to reconstruct the historical scenario of colonization of such a lake and evaluated the osmoregulatory shift associated to changes in habitat and life cycle between amphidromous and landlocked populations., Results: Standard diversity indices including the average number of nucleotide differences (Π) and the haplotype diversity index (H) indicated that both populations were, as expected, genetically distinctive, being the landlocked population less diverse than the diadromous one. Similarly, pairwise GST and NST comparison detected statistically significant differences between both populations, while genealogy of haplotypes evidenced a recent founder effect from the diadromous stock, followed by an expansion process in the lake. To test for physiological differences, individuals of both populations were challenged with a range of salinities from 0 to 30 ppt for 8 days following a period of progressive acclimation. The results showed that the landlocked population had a surprisingly wider tolerance to salinity, as landlocked fish survival was 100% from 0 to 20 ppt, whereas diadromous fish survival was 100% only from 10 to 15 ppt. The activity of ATPase enzymes, including Na+/K+-ATPase (NKA), and H+-ATPase (HA) was measured in gills and intestine. Activity differences were detected between the populations at the lowest salinities, including differences in ATPases other than NKA and HA. Population differences in mortality are not reflected in enzyme activity differences, suggesting divergence in other processes., Conclusions: These results clearly demonstrate the striking adaptive changes of G. maculatus osmoregulatory system, especially at hyposmotic environments, associated to a drastic shift in habitat and life cycle at a scale of a few thousand years.

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.

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 HCO 3 – 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 ~ 1700 nmol HCO 3 – 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. [ABSTRACT FROM AUTHOR]

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 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (1.25mg 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. • AQUI-S® resulted quite effective for handling procedures in seabream juveniles. • Etomidate was completely ineffective for handling procedures in seabream juveniles. • AQUI-S® (2 ppm of isoeugenol) for seabream transport increased plasma secondary stress responses. • Etomidate (0.15 ppm) for seabream transport reduced energy expenditure in liver. • AQUI-S® and etomidate reduced ion-loss through the gills during transport. [ABSTRACT FROM AUTHOR]

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. • This is the first study on the effect of stocking density on Eleginops maclovinus. • Stocking density modulates the physiology of Eleginops maclovinus. • High stocking density over the long-term generates low levels of cortisol and tissue-specific metabolic modulation. • We recommend to cultivate Eleginops maclovinus in the range of 3–12 kg/m3, avoiding a high density of 24 kg/m3. [ABSTRACT FROM AUTHOR]