An in vitro analysis of intestinal ammonia handling in fasted and fed freshwater rainbow trout (Oncorhynchus mykiss)

Ammonia transport and metabolism were investigated in the intestinal tract of freshwater rainbow trout which had been either fasted for 7 days, or fasted then fed a satiating meal of commercial trout pellets. In vivo, total ammonia concentrations (T _amm) in the chyme were approximately 1 mmol L^?1 across the entire intestine at 24 h after the meal. Highest chyme pH and P _NH3 values occurred in the posterior intestine. In vitro gut sac experiments examined ammonia handling with mucosal (Jm_amm) and serosal (Js_amm) fluxes under conditions of fasting and feeding, with either background (control ≤0.013 mmol L^?1) or high luminal ammonia concentrations (HLA = 1 mmol L^?1), the latter mimicking those seen in chyme in vivo. Feeding status (fasted or fed) appeared to influence ammonia handling by each individual section. The anterior intestine exhibited the greatest Jm_amm and Js_amm values under fasted control conditions, but these differences tended to disappear under typical post-feeding conditions when total endogenous ammonia production (Jt_amm = Js_amm ? Jm_amm, signs considered) was greatly elevated in all intestinal sections. Under fasted conditions, glutamate dehydrogenase (GDH) and glutaminase (GLN) activities were equal across all sections, but the ammonia-trapping enzyme glutamine synthetase (GS) exhibited highest activity in the posterior intestine, in contradiction to previous literature. Feeding clearly stimulated the total rate of endogenous ammonia production (Jt_amm), even in the absence of a high luminal ammonia load. This was accompanied by an increase in GDH activity of the anterior intestine, which was also the site of the largest Jt_amm. In all sections, during HLA exposure, either alone or in combination with feeding, there were much larger increases in endogenous Jt_amm, most of which was effluxed to the serosal solution. This is interpreted as a response to avoid potential cytotoxicity due to overburdened detoxification mechanisms in the face of elevated mucosal ammonia. Thus T _amm of the intestinal tissue remained relatively constant regardless of feeding status and exposure to HLA. Ammonia production by the gut may explain up to 18 % of whole-body ammonia excretion in vivo under fasting conditions, and 47 % after feeding, of which more than half originates from endogenous production rather than from absorption from the lumen.     

What is the primary function of the early teleost gill? Evidence for Na+/NH+4 exchange in developing rainbow trout (Oncorhynchus mykiss)

Post-hatch fishes lack a functional gill and use cutaneous surfaces for exchange with the surrounding environment. The ionoregulatory hypothesis posits that ionoregulation is the first physiological process to be limited by cutaneous exchange, necessitating its shift to the gills. We hypothesized that the ontogeny of branchial ammonia excretion (J_amm) is coupled to Na⁺ uptake () in accordance with the current model for exchange in freshwater. Using divided chambers, branchial and cutaneous J_amm, and oxygen consumption (MO₂) by larval rainbow trout were assessed. Following hatch, the skin accounted for 97% and 86% of total J_amm and , respectively. J_amm and shifted to the gills simultaneously at 15 days post-hatch (dph) and were highly correlated (R² = 0.951) at the gills, but not the skin, over development. Contrastingly, MO₂ shifted significantly later at 27 dph, in agreement with the ionoregulatory hypothesis. Moreover, the mRNA expression and/or enzymatic activity of Rhesus proteins, Na⁺/H⁺-exchanger, H⁺-ATPase, Na⁺/K⁺-ATPase and carbonic anhydrase, all key components of the -exchange system, increased in the gills over larval development. We propose that the ontogeny of branchial occurs as exchange and provide evidence for a novel element to the ionoregulatory hypothesis, the excretion of potentially lethal metabolic ammonia.     

Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na⁺/K⁺-ATPase, H⁺-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC₅₀ value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na⁺/NH₄⁺-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H⁺-ATPase and Na⁺/K⁺-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H⁺-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H⁺-ATPase and Rhcg1 to the similar non-Na⁺/K⁺-ATPase immunoreactive cell type would support a role for H⁺-ATPase in ammonia excretion via Rhcg by NH₄⁺ trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H⁺-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H⁺-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH₃ permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.     

Physiological and molecular analysis of the interactive effects of feeding and high environmental ammonia on branchial ammonia excretion and Na+ uptake in freshwater rainbow trout

Recently, a “Na⁺/NH₄ ⁺ exchange complex” model has been proposed for ammonia excretion in freshwater fish. The model suggests that ammonia transport occurs via Rhesus (Rh) glycoproteins and is facilitated by gill boundary layer acidification attributable to the hydration of CO₂ and H⁺ efflux by Na⁺/H⁺ exchanger (NHE-2) and H⁺-ATPase. The latter two mechanisms of boundary layer acidification would occur in conjunction with Na⁺ influx (through a Na⁺ channel energized by H⁺-ATPase and directly via NHE-2). Here, we show that natural ammonia loading via feeding increases branchial mRNA expression of Rh genes, NHE-2, and H⁺-ATPase, as well as H⁺-ATPase activity in juvenile trout, similar to previous findings with ammonium salt infusions and high environmental ammonia (HEA) exposure. The associated increase in ammonia excretion occurs in conjunction with a fourfold increase in Na⁺ influx after a meal. When exposed to HEA (1.5 mmol/l NH₄HCO₃ at pH 8.0), both unfed and fed trout showed differential increases in mRNA expression of Rhcg2, NHE-2, and H⁺-ATPase, but H⁺-ATPase activity remained at control levels. Unfed fish exposed to HEA displayed a characteristic reversal of ammonia excretion, initially uptaking ammonia, whereas fed fish (4 h after the meal) did not show this reversal, being able to immediately excrete ammonia against the gradient imposed by HEA. Exposure to HEA also led to a depression of Na⁺ influx, demonstrating that ammonia excretion can be uncoupled from Na⁺ influx. We suggest that the efflux of H⁺, rather than Na⁺ influx itself, is critical to the facilitation of ammonia excretion.     

The skin of adult rainbow trout is not a significant site of ammonia clearance from the blood

We hypothesized that the skin acts as an extrabranchial route for ammonia excretion in adult rainbow trout (Oncorhynchus mykiss) following high environmental ammonia (HEA) exposure. Trunks of control or HEA-exposed trout were perfused with saline containing 0 or 1 mmol l⁻¹ NH₄⁺. Cutaneous ammonia excretion rates increased 2.5-fold following HEA exposure, however there was no difference in rates between trunks perfused with 0 or 1 mmol l⁻¹ NH₄⁺. The skin is therefore capable of excreting its own ammonia load, but it does not clear circulating ammonia from the plasma.     

Anti-Oxidative Defences Are Modulated Differentially in Three Freshwater Teleosts in Response to Ammonia-Induced Oxidative Stress

Oxidative stress and the antioxidant response induced by high environmental ammonia (HEA) were investigated in the liver and gills of three freshwater teleosts differing in their sensitivities to ammonia. The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed to 1 mM ammonia (as NH₄HCO₃) for 0 h (control), 3 h, 12 h, 24 h, 48 h, 84 h and 180 h. Results show that HEA exposure increased ammonia accumulation significantly in the liver of all the three fish species from 24 h–48 h onwards which was associated with an increment in oxidative stress, evidenced by elevation of xanthine oxidase activity and levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Unlike in trout, H₂O₂ and MDA accumulation in carp and goldfish liver was restored to control levels (84 h–180 h); which was accompanied by a concomitant increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase activity and reduced ascorbate content. Many of these defence parameters remained unaffected in trout liver, while components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase) enhanced to a greater extent. The present findings suggest that trout rely mainly on glutathione dependent defensive mechanism while carp utilize SOD, CAT and ascorbate as anti-oxidative sentinels. Hepatic cells of goldfish appear to utilize each of these protective systems, and showed more effective anti-oxidative compensatory responses towards HEA than carp, while trout were least effective. The present work also indicates that HEA exposure resulted in a relatively mild oxidative stress in the gills of all three species. This probably explains the almost complete lack of anti-oxidative responses in branchial tissue. This research suggests that oxidative stress, as well as the antioxidant potential clearly differ between salmonid and cyprinid species.     

Mechanisms of Na+ uptake,ammonia excretion,and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi,Hemigrammus rhodostomus,and Moenkhausia diktyota)

Mechanisms of Na⁺ uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J _in), efflux (J _out), and net flux rates (J _net) of Na⁺ and Cl^?, and the net excretion rate of ammonia (J _Amm). The fish were collected directly from the Rio Negro, and studied in their native “blackwater” which is acidic (pH 4.5), ion-poor (Na⁺, Cl^? ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l^?1). J _in ^Na , J _in ^Cl , and J _Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J _in ^Na was unaffected by amiloride (10^?4 M, NHE and Na⁺ channel blocker), but both J _in ^Na and J _in ^Cl were virtually eliminated (85–99 % blockade) by AgNO₃ (10^?7 M). A time course study on cardinal tetras demonstrated that J _in ^Na blockade by AgNO₃ was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10^?4 M) caused a 50 % reduction in J _in ^Na _.. Additionally, J _in ^Na was unaffected by phenamil (10^?5 M, Na⁺ channel blocker), bumetanide (10^?4 M, NKCC blocker), hydrochlorothiazide (5 × 10^?3 M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J _in ^Na (by acetazolamide and AgNO₃ respectively), had any inhibitory effect on J _Amm. Therefore, Na⁺ uptake in Rio Negro tetras depends on an internal supply of H⁺ from CA, but does not fit any of the currently accepted H⁺-dependent models (NHE, Na⁺ channel/V-type H⁺-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current “Na⁺/NH₄ ⁺ exchange metabolon” paradigm. Na⁺, K⁺-ATPase and V-type H⁺-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH₄Cl, 10^?3 M) significantly increased J _in ^Na , and NH₄ ⁺ was equally or more effective than K⁺ in activating branchial Na⁺,(K⁺) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na⁺ uptake, but that Na⁺ uptake (by an as yet unknown H⁺-dependent apical mechanism) depends on ammonia excretion, driven by active NH₄ ⁺ entry via basolateral Na⁺,(K⁺)-ATPase.     

Immunohistochemical localization of urea and ammonia transporters in two confamilial fish species, the ureotelic gulf toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus)

This study aims to illustrate potential transport mechanisms behind the divergent approaches to nitrogen excretion seen in the ureotelic toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus). Specifically, we wish to confirm the expression of a urea transporter (UT), which is found in the gill of the toadfish and which is responsible for the unique “pulsing” nature of urea excretion and to localize the transporter within specific gill cells and at specific cellular locations. Additionally, the localization of ammonia transporters (Rhesus glycoproteins; Rhs) within the gill of both the toadfish and midshipman was explored. Toadfish UT (tUT) was found within Na⁺-K⁺-ATPase (NKA)-enriched cells, i.e., ionocytes (probably mitochondria-rich cells), especially along the basolateral membrane and potentially on the apical membrane. In contrast, midshipman UT (pnUT) immunoreactivity did not colocalize with NKA immunoreactivity and was not found along the filaments but instead within the lamellae. The cellular location of Rh proteins was also dissimilar between the two fish species. In toadfish gills, the Rh isoform Rhcg1 was expressed in both NKA-reactive cells and non-reactive cells, whereas Rhbg and Rhcg2 were only expressed in the latter. In contrast, Rhbg, Rhcg1 and Rhcg2 were expressed in both NKA-reactive and non-reactive cells of midshipman gills. In an additional transport epithelium, namely the intestine, the expression of both UTs and Rhs was similar between the two species, with only subtle differences being observed.     

The effects of extremely alkaline water (pH 9·5) on rainbow trout gill function and morphology

Rainbow trout that were held under control conditions, at pH8·0, in moderately hard Hamilton tap water, had Cl^? and Na⁺ influx rates (J^CLin and J^Na, respectively) of 270 and 300 μmol kg^?1 h^?1, respectively. Exposure to pH 9·5 water led to an immediate 67% decline in J^CL_in and a 45% reduction in J^Na_in at 0–1 h. Influx rates declined further and by 4–5 h the net decreases in both J^CL_in and J^Na_in approximated 80%. By 24 h J^CL_in had recovered to rates not significantly different from those at pH 8·0; while J^Na_in only partially recovered and remained about 50% lower than control measurements through 72 h. The complete recovery of J^CL_in and partial recovery of J^Na_in may have been related to a fourfold greater branchial chloride cell (CC) fractional surface area observed in rainbow trout exposed to pH 9·5 for 72 h. Ammonia excretion (J_Amm) was about 170 μmol N kg^?1 h^?1 at pH 8·0 but was initially reduced by 90% over the first hour of high pH exposure. J_Amm rapidly recovered and by 24 h it had returned to pre-exposure levels. This recovery tended to parallel the partial recovery of J^Na_in. However, subsequent addition of amiloride (10^?4M) to the water at 75 h led to no change in J_Amm, despite a 50% reduction in J^Na_in. Thus, it does not appear that there is a linkage between Na⁺ influx and the recovery of ammonia excretion under highly alkaline conditions.     

Ammonia (C-Methylamine) Transport across the Bacteroid and Peribacteroid Membranes of Soybean Root Nodules

[¹⁴C]Methylamine (MA; an analog of ammonia) was used to investigate ammonia transport across the bacteroid and peribacteroid membranes (PBM) from soybean (Glycine max) root nodules. Free-living Bradyrhizobium japonicum USDA110 grown under nitrogen-limited conditions showed rapid MA uptake with saturation kinetics at neutral pH, indicative of a carrier. Exchange of accumulated MA for added ammonia occurred, showing that the carrier recognized both NH₄⁺ and CH₃NH₃⁺. MA uptake by isolated bacteroids, on the other hand, was very slow at low concentrations of MA and increased linearly with increasing MA concentration up to 1 millimolar. Ammonia did not inhibit MA by isolated bacteroids and did not cause efflux of accumulated MA. PBM-enclosed bacteroids (peribacteroid units [PBUs]) were qualitatively similar to free bacteroids with respect to MA transport. The rates of uptake and efflux of MA by PBUs were linearly dependent on the imposed concentration gradient and unaffected by NH₄Cl. MA uptake by PBUs increased exponentially with increasing pH, confirming that the rate increased linearly with increasing CH₃NH₂ concentration. The results are consistent with other evidence that transfer of ammonia from the nitrogen-fixing bacteroid to the host cytosol in soybean root nodules occurs solely by simple diffusion of NH₃ across both the bacteroid and peribacteroid membranes.     

Sodium Efflux and Potential Differences across the Irrigated Gill of Sea Water-Adapted Rainbow Trout (Salmo gairdneri)

Sodium extrusion (J_out^Na) was measured across the gills of rainbow trout, Salmo gairdneri, adapted to sea water (SW) using a gill-irrigation system of small volume. The potential difference (TEP) was also measured under similar conditions. J_out^Na was usually between 100–250 µeq (100 g)^–1 h^–1, about an order of magnitude faster than in fresh water (FW)-adapted trout, but slower than has been reported for any other marine teleost. The TEP was between 10–11 mV, body fluids positive to SW. When the external medium was changed from SW to FW J_out^Na was reduced to about 25 % of the initial value, and the TEP was reduced by 40–50 mV (i.e. body fluids negative by 30–40 mV). Addition of either Na⁺ or K⁺ in SW concentrations reversed the changes; J_out^Na increased and the gill repolarized. The electrical behavior and sodium efflux in irrigated trout gill is qualitatively the same as has been reported for unanaesthetized, free-swimming fish of other species. Thus, the irrigated gill provides an adequate model for studying the mechanism of sodium extrusion in marine teleosts.     

Age and growth of Siberian sculpin (Cottus poecilopus) and young brown trout (Salmo trutta) in a subalpine Norwegian river

Age, growth and density of Siberian sculpin (Cottus poecilopus) and young brown trout (Salmo trutta) within two sections of River Atna; above Lake Atnsjøen [Section 1 at altitudes between 739 and 715 m] and below Lake Atnsjøen [Section 2 at altitudes between 430 and 370 m] was studied during a 6-year period (1986–91). The water temperature was considerably lower in Section 1 than in Section 2, as the number of days with a water temperature above 10?°C (T _{D ≥ 10?° C} ) from spring to August 1 ranged between 2–26 and 26–52 days, respectively. Juvenile brown trout (age 0+) attained a significantly smaller body size in Section 1 than in Section 2; mean length ±SD was 35 ± 8 mm (ranged 27–46) and 43 ± 7 mm (range 38–46), respectively. In Section 2, there was a highly positive correlation between the body length of 0+ brown trout and mean water temperature in June (p<0.005), and also to some extent in Section 1 (p=0.11). Individuals of age 1+ did not exhibit any such difference, while fish in age group 2+ were larger in Section 1 than in Section 2. By using the number of days with a water temperature between the range 5–10?°C (T _{D ≥ 5 ? 10?° C} ) as test variables, we found a highly positive correlation between the August 1 body length of 0+ brown trout and T _{D ≥ 9}?°C from spring to August 1 in Section 2 (p<0.05), as opposed to T _{D ≥ 7}?°C for trout in Section 1 (p=0.11). Young Siberian sculpin (age 0+ and 1+) also exhibited slower growth in Section 1 than in Section 2, but this was not the case among older specimens. In the year with the lowest temperature measured (1987), no 0+ Siberian sculpin were caught in any of the two sections, indicating that low temperature affects their survival. Both species exhibited large spatial and temporal variation in density. Thus, data on abundance and growth sampled on one occasion at one site can not be regarded as representative for these two fish populations.     

Environmental and anthropogenic correlates of hybridization between westslope cutthroat trout (Oncorhynchus clarkii lewisi) and introduced rainbow trout (O. mykiss)

Hybridization with introduced taxa is one of the major threats to the persistence of native biodiversity. The westslope cutthroat trout (Oncorhynchus clarkii lewisi) is found in southeastern British Columbia and southwestern Alberta, Canada, and adjacent areas of Montana, Idaho, and Washington State, USA. Through much of this area, native populations are threatened by hybridization with introduced rainbow trout (O. mykiss). We surveyed 159 samples comprising over 5,000 fish at 10 microsatellite DNA loci to assess the level of admixture between native westslope cutthroat trout (wsct) and introduced rainbow trout in southwestern Alberta. Admixture levels (q_wsct of 0 = pure rainbow trout, q_wsct of 1.0 = pure westslope cutthroat trout) ranged from <0.01 to 0.99 and averaged from 0.72 to 0.99 across seven drainage areas. Regression tree analyses indicated that water temperature, elevation, distance to the nearest stocking site, and distance to the nearest railway line were significant components of a model that explained 34 % of the variation across sites in q_wsct across 58 localities for which habitat variables were available. Partial dependence plots indicated that admixture with rainbow trout increased with increasing water temperature and distance to the nearest railway line, but decreased with increasing elevation and distance from stocking site to sample site. Our results support the hypothesis that westslope cutthroat trout may be less susceptible to hybridization with rainbow trout in colder, higher elevation streams, and illustrate the interaction between abiotic and anthropogenic factors in influencing hybridization between native and introduced taxa.     

Comparing competitive ability and associated metabolic traits between a resident and migratory population of bull trout against a non-native species

Juvenile bull trout Salvelinus confluentus from two geographically and ecologically distinct populations were compared with regard to their ability to compete with non-native brook trout Salvelinus fontinalis in an artificial stream, and with respect to their rates of oxygen consumption. Bull trout collected from a migratory population foraged more successfully against brook trout competitors than those from a resident population, capturing more of a limited amount of food items presented. The migratory population was also more aggressive (measured by the number of nips, chases and lateral threat displays) against brook trout competitors than the resident population. Bull trout from the migratory population had a higher oxygen consumption rate (203 mg O₂ kg · hr^-1) in the field than similar sized fish from the resident population (183 mg O₂ kg · hr^-1). These results suggest native bull trout have population-level variation in competitive ability against a non-native species and such competitive ability is positively associated with metabolism and migratory life history.     

Annual variability in the life-history characteristics of brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus) in a subalpine Norwegian lake

The annual variability in growth and life history traits of brown trout (Salmo trutta L.) and Arctic charr (Salvelinus alpinus (L.)) in Lake Atnsjøen, a Norwegian subalpine lake, was studied over a period of 13 years (1985–1997). The extent to which life-history characteristics recorded on one occasion can be regarded as representative for the population was explored. We found inter-cohort variation in growth for both species; estimates of asymptotic length (L _∞) in ten cohorts ranged between 225–305 mm (CV = 10.5%) for brown trout and 273–301 mm (CV = 4.1%) for Arctic charr. However, this variation was much lower than inter-population variation for brown trout based on single samples from 169 populations (CV = 24.6%). In Lake Atnsjøen, annual growth increment correlated highly with the number of days warmer than 7?°C (R ²=0.60–0.89) for brown trout, and days warmer than 10?°C (R ²=0.40–0.58) for Arctic charr. Females of Arctic charr were younger at sexual maturity than males, while no such difference was found in brown trout. Generally speaking, early maturing individuals of both species grew faster, particularly from age-2 and onwards, than immature individuals. Early maturing individuals, however, were smaller at maturity than those maturing one year older. Age and size at maturity were significantly correlated with asymptotic lengths only in Arctic charr females.     

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.     

Discontinuous ammonia excretion and glutamine storage in littoral Oniscidea (Crustacea: Isopoda): testing tidal and circadian models

A key evolutionary development facilitating land colonization in terrestrial isopods (Isopoda: Oniscidea) is the intermittent liberation of waste nitrogen as volatile ammonia. Intermittent ammonia release exploits glutamine (Gln) as an intermediary nitrogen store. Here, we explore the relationship between temporal patterns of ammonia release and Gln accumulation in three littoral oniscideans from Southern California. Results are interpreted in terms of water availability, habitat, activity patterns, and ancestry. A two-way experimental design was used to test whether ammonia excretion and Gln accumulation follow a tidal or diel periodicity. Ammonia excretion was studied in the laboratory using chambers with or without available seawater and using an acid trap to collect volatile ammonia. Ligia occidentalis releases ammonia directly into seawater and accumulates Gln during low tide (48.9 ± 6.5 μmol g^?1 at low tide, 24.1 ± 3.0 μmol g^?1 at high tide), indicating that excretion is tidally constrained. Alloniscus perconvexus and Tylos punctatus can excrete ammonia directly into seawater or utilize volatilization. Both species burrow in sand by day and show a diel excretory pattern, accumulating Gln nocturnally (31.8 ± 2.7 μmol g^?1 at dawn and 21.8 ± 2.3 μmol g^?1 at dusk for A. perconvexus; 85.7 ± 15.1 μmol g^?1 at dawn and 25.4 ± 2.9 μmol g^?1 at dusk for T. punctatus) and liberating ammonia diurnally. Glutaminase shows higher activity in terrestrial (0.54–0.86 U g^?1) compared to intertidal (0.25–0.31 U g^?1) species, consistent with the need to generate high PNH₃ for volatilization. The predominant isoform in Armadillidium vulgare is phosphate dependent and maleate independent; phosphate is a plausible regulator in vivo.     

Effects of dietary fibre and protein on urea transport across the cecal mucosa of piglets

In ruminants, gastrointestinal recycling of urea is acutely enhanced by fibre-rich diets that lead to high ruminal concentration of short chain fatty acids (SCFA), while high ammonia has inhibitory effects. This study attempted to clarify if urea flux to the porcine cecum is similarly regulated. Thirty-two weaned piglets were fed diets containing protein (P) of poor prececal digestibility and fibre (F) at high (H) or low levels (L) in a 2 × 2 factorial design. After slaughter, cecal content was analyzed and the cecal mucosa incubated in Ussing chambers to measure the effect of pH, SCFA and NH₄ ⁺ on the flux rates of urea, short-circuit current (I _sc) and tissue conductance (G _t). NH₄ ⁺ significantly enhanced I _sc (from 0.5 ± 0.2 to 1.2 ± 0.1 μEq cm^?2 h^?1). No acute effects of SCFA or ammonia on urea flux were observed. Tissue conductance was significantly lower in the high dietary fibre groups irrespective of the protein content. Only the HP-LF group emerged as different from all others in terms of urea flux (74 ± 6 versus 53 ± 3 nmol cm^?2 h^?1), associated with higher cecal ammonia concentration and reduced fecal consistency. The data suggest that as in the rumen, uptake of ammonia by the cecum may involve electrogenic transport of the ionic form (NH₄ ⁺). In contrast to findings in the rumen, neither a high fibre diet nor acute addition of SCFA enhanced urea transport across the pig cecum. Instead, a HP-LF diet had stimulatory effects. A potential role for urea recycling in stabilizing luminal pH is discussed.     

Induction of salt tolerance in Azolla microphylla Kaulf through modulation of antioxidant enzymes and ion transport

Azolla microphylla plants exposed directly to NaCl (13 dsm^-1) did not survive the salinity treatment beyond a period of one day, whereas plants exposed directly to 4 and 9 dsm^-1 NaCl were able to grow and produce biomass. However, plants pre-exposed to NaCl (2 dsm^-1) for 7 days on subsequent exposure to 13 dsm^-1 NaCl were able to grow and produce biomass although at a slow rate and are hereinafter designated as pre-exposed plants. The pre-exposed and directly exposed plants distinctly differed in their response to salt in terms of lipid peroxidation, proline accumulation, activity of antioxidant enzymes, such as SOD, APX, and CAT, and Na⁺/K⁺ ratio. Efficient modulation of antioxidant enzymes coupled with regulation of ion transport play an important role in the induction of salt tolerance. Results show that it is possible to induce salt adaptation in A. microphylla by pre-exposing them to low concentrations of NaCl.