Nutritional Status as the Key Modulator of Antioxidant Responses Induced by High Environmental Ammonia and Salinity Stress in European Sea Bass (Dicentrarchus labrax).

Salinity fluctuation is one of the main factors affecting the overall fitness of marine fish. In addition, water borne ammonia may occur simultaneously with salinity stress. Additionally, under such stressful circumstances, fish may encounter food deprivation. The physiological and ion-osmo regulatory adaptive capacities to cope with all these stressors alone or in combination are extensively addressed in fish. To date, studies revealing the modulation of antioxidant potential as compensatory response to multiple stressors are rather lacking. Therefore, the present work evaluated the individual and combined effects of salinity challenge, ammonia toxicity and nutritional status on oxidative stress and antioxidant status in a marine teleost, European sea bass (Dicentrarchus labrax). Fish were acclimated to normal seawater (32 ppt), to brackish water (20 ppt and 10 ppt) and to hypo-saline water (2.5 ppt). Following acclimation to different salinities for two weeks, fish were exposed to high environmental ammonia (HEA, 20 mg/L representing 50% of 96h LC50 value for ammonia) for 12 h, 48 h, 84 h and 180 h, and were either fed (2% body weight) or fasted (unfed for 7 days prior to HEA exposure). Results show that in response to decreasing salinities, oxidative stress indices such as xanthine oxidase activity, levels of hydrogen peroxide (H2O2) and lipid peroxidation (malondialdehyde, MDA) increased in the hepatic tissue of fasted fish but remained unaffected in fed fish. HEA exposure at normal salinity (32 ppt) and at reduced salinities (20 ppt and 10 ppt) increased ammonia accumulation significantly (84 h-180 h) in both feeding regimes which was associated with an increment of H2O2 and MDA contents. Unlike in fasted fish, H2O2 and MDA levels in fed fish were restored to control levels (84 h-180 h); with a concomitant increase in superoxide dismutase (SOD), catalase (CAT), components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase), ascorbate peroxidase (APX) activity and reduced ascorbate (ASC) content. On the contrary, fasted fish could not activate many of these protective systems and rely mainly on CAT and ASC dependent pathways as antioxidative sentinels. The present findings exemplify that in fed fish single factors and a combination of HEA exposure and reduced seawater salinities (upto 10 ppt) were insufficient to cause oxidative damage due to the highly competent antioxidant system compared to fasted fish. However, the impact of HEA exposure at a hypo-saline environment (2.5 ppt) also defied antioxidant defence system in fed fish, suggesting this combined factor is beyond the tolerance range for both feeding groups. Overall, our results indicate that the oxidative stress mediated by the experimental conditions were exacerbated during starvation, and also suggest that feed deprivation particularly at reduced seawater salinities can instigate fish more susceptible to ammonia toxicity.

Interactive effect of high environmental ammonia and nutritional status on ecophysiological performance of European sea bass (Dicentrarchus labrax) acclimated to reduced seawater salinities.

We investigated the interactive effect of ammonia toxicity, salinity challenge and nutritional status on the ecophysiological performance of European sea bass (Dicentrarchus labrax). Fish were progressively acclimated to normal seawater (32ppt), to brackish water (20ppt and 10ppt) and to hyposaline water (2.5ppt). Following acclimation to different salinities for two weeks, fish were exposed to high environmental ammonia (HEA, 20mg/L ∼1.18mM representing 50% of 96h LC50 value for ammonia) for 12h, 48h, 84h and 180h, and were either fed (2% body weight) or fasted (unfed for 7 days prior to HEA exposure). Biochemical responses such as ammonia (Jamm) and urea excretion rate, plasma ammonia, urea and lactate, plasma ions (Na(+), Cl(-) and K(+)) and osmolality, muscle water content (MWC) and liver and muscle energy budget (glycogen, lipid and protein), as well as branchial Na(+)/K(+)-ATPase (NKA) and H(+)-ATPase activity, and branchial mRNA expression of NKA and Na(+)/K(+)/2Cl(-) co-transporter (NKCC1) were investigated in order to understand metabolic and ion- osmoregulatory consequences of the experimental conditions. During HEA, Jamm was inhibited in fasted fish at 10ppt, while fed fish were still able to excrete efficiently. At 2.5ppt, both feeding groups subjected to HEA experienced severe reductions and eventually a reversion in Jamm. Overall, the build-up of plasma ammonia in HEA exposed fed fish was much lower than fasted ones. Unlike fasted fish, fed fish acclimated to lower salinities (10ppt-2.5ppt) could maintain plasma osmolality, [Na(+)], [Cl(-)] and MWC during HEA exposure. Thus fed fish were able to sustain ion-osmotic homeostasis which was associated with a more pronounced up-regulation in NKA expression and activity. At 2.5ppt both feeding groups activated H(+)-ATPase. The expression of NKCC1 was down-regulated at lower salinities in both fed and fasted fish, but was upregulated within each salinity after a few days of HEA exposure. Though an increment in plasma lactate content and a decline in energy stores were noted for both feeding regimes, the effect was more severe in feed deprived fish. Overall, several different physiological processes were disturbed in fasted sea bass during HEA exposure while feeding alleviated adverse effects of high ammonia and salinity challenge. This suggests that low food availability can render fish more vulnerable to external ammonia, especially at reduced seawater salinities.