Ammonia Efflux Research Articles

Effects of carbonate alkalinity on branchial gene expression in the large-scale loach (Paramisgurnus dabryanus)

Elucidating the mechanisms of alkaline tolerance in freshwater teleosts will help in the development of commercial saline-alkaline aquaculture. The large-scale loach (Paramisgurnus dabryanus) is a viable species for such aquaculture, but the mechanisms of its tolerance of alkaline water are unclear. Large-scale loach was exposed to 40, 50, and 60 mmol L-1 NaHCO3 for 12, 48, and 96 h to evaluate the transcriptional changes of branchial Rhesus (Rh) glycoproteins, and aquaporins (Aqp)1 and Aqp3. Rhag transcript levels increased with longer exposure times. Rhag expression also rose considerably at higher carbonate alkalinities. Rhbg mRNA levels declined significantly under carbonate alkalinity exposure. A marked up-regulation of Rhcg was observed in the gills of the loach. Exposure to 60 mmol L-1 NaHCO3 also induced a significant up-regulation of aqp1. By contrast, aqp3 expression was significantly lower after 48 h exposure. The current findings reveal that the large-scale loach up-regulates Rhag and Rhcg to enhance ammonia efflux from the gills when exposed to high alkalinity. It is proposed that this species maintains appropriate osmolality when adapting to an alkaline environment by down-regulating aqp3 (to impede urea removal) and up-regulating aqp1 in the gills (to excrete excessive internal water).

Scaling-up ecosystem functions of coastal heterogeneous sediments: testing practices using high resolution data

ContextHeterogeneity in coastal soft sediments and the difficulty of data collection hinder our ability to scale up ecological data (necessarily obtained at small-scale) to large-scale. The use of scaling in marine ecology is not as common as in terrestrial ecology and current practices are often too simplistic and inadequate.ObjectivesWe aimed to demonstrate that the use of different scaling approaches leads to considerably different results and that not accounting for ecological heterogeneity decreases our ability to accurately extrapolate measurements of ecosystem functions performed by intertidal soft sediment habitats.MethodsHigh resolution raster maps of sediment denitrification, ammonia (NH4+) efflux and organic matter degradation were sampled to produce a simulated dataset and compare the performance of three different scaling approaches: direct scaling, spatial allometry and semivariogram/kriging.ResultsDirect scaling underestimated denitrification, NH4+ efflux and organic matter degradation (84.1, 84.9 and 90.3% less) while allometry underestimated denitrification (81.9% less) but overestimated NH4+ efflux and organic matter degradation (2594.1 and 14,879.9% more). Kriging produced more accurate results and the predicted functions only differed from the estimated values by 14.7, 29.4 and 3.9% respectively.ConclusionsOur work shows that the choice of the scaling method is crucial in estimating intertidal soft sediment functions and highlights the need for empirical and theoretical models that link ecosystem functioning to biological attributes that can be measured remotely over large areas. Integrating measures of heterogeneity through the spatial structure of the data leads to outcomes that are more realistic and relevant to resource management.

Physio-biochemical, metabolic nitrogen excretion and ion-regulatory assessment in largemouth bass (Micropterus salmoides) following exposure to high environmental iron

Iron overload is a significant water quality issue in many parts of the world. Therefore, we evaluated the potential toxic effects of waterborne elevated iron on largemouth bass (Micropterus salmoides), a highly valued sport and aquaculture fish species. First, a 96 h-LC50 toxicity assay was performed to understand the tolerance limit of this species to iron; and was determined to be 22.07 mg/L (as Fe3+). Thereafter, to get a better insight on the fish survival during long-term exposure to high environmental iron (HEI) (5.52 mg/L, 25% of the determined 96 h-LC50 value), a suite of physio-biochemical, nitrogenous metabolic and ion-regulatory compensatory responses were examined at 7, 14, 21 and 28 days. Results showed that oxygen consumption dropped significantly at 21 and 28 days of HEI exposure. Ammonia excretion rate (Jamm) was significantly inhibited from day 14 and remained suppressed until the last exposure period. The transcript concentration of Rhesus glycoproteins Rhcg2 declined; likely diminishing ammonia efflux out of gills. These changes were also reflected by a parallel increment in plasma ammonia levels. Under HEI exposure, ion-balance was negatively affected, manifested by reduced plasma [Na+] and parallel inhibition in branchial Na+/K+-ATPase activity. Muscle water content was elevated in HEI-exposed fish, signifying an osmo-regulatory compromise. HEI exposure also increased iron burden in plasma and gills. The iron accumulation pattern in gills was significantly correlated with a suppression of Jamm, branchial Rhcg2 expression and Na+/K+-ATPase activity. There was also a decline in the glycogen, protein and lipid reserves in the hepatic tissue from 14 days, 28 days and 21 days, respectively. Overall, we conclude that sub-lethal chronic iron exposure can impair normal physio-biochemical and ion-regulatory functions in largemouth bass. Moreover, this data set can be applied in assessing the environmental risk posed by a waterborne iron overload on aquatic life.

Combined effects of waterborne copper exposure and salinity on enzymes related to osmoregulation and ammonia excretion by blue crab Callinectes sapidus.

Copper is essential, but can be toxic to aquatic organisms when present in high concentrations. In freshwater crustaceans, copper inhibits enzymes related to ionic and osmoregulation and to the ammonia efflux, that leads to Na+ imbalance and inhibition of ammonia excretion. In the animals inhabiting estuarine or seawater, mechanisms of copper toxicity is not clear, but had been described as disruption of ionregulation and metabolism. To clarify the mechanism of copper toxicity in crustaceans inhabiting variable salinity, this work investigated whether copper affects ammonia excretion and enzymes used for ammonia balance and osmoregulation in the blue crab Callintectes sapidus acclimated to salinity 2 and 30 ppt. To achieve this, juveniles of the blue crab were exposed to 63.5 µg/L of copper at both salinities for 96 h. This is an environmentally realistic copper concentration. Results of ammonia efflux, free amino acids and Na+ concentrations in hemolymph, Na+/K+-ATPase, H+-ATPase and, carbonic anhydrase (CA) activities in gills were consistent with the osmoregulatory pattern adopted by the blue crab, which hyperosmoregulates at salinity 2 ppt and osmoconforms at 30 ppt. At 30 ppt copper reduced free amino acid in hemolymph of crabs, suggesting an effect of the metal on osmotic performance. At 2 ppt, copper significantly increased the H+-ATPase activity involved in ammonia excretion. This may be a compensatory response of crabs to maintain low levels of ammonia in their hemolymph; which can be increased by copper exposure. Results presented here are useful for the improvement of the Biotic Ligand Model (BLM) to predict copper toxicity for saltwater environments.

Pre-acclimation to low ammonia improves ammonia handling in common carp (Cyprinus carpio) when exposed subsequently to high environmental ammonia

We tested whether exposing fish to low ammonia concentrations induced acclimation processes and helped fish to tolerate subsequent (sub)lethal ammonia exposure by activating ammonia excretory pathways. Common carp (Cyprinus carpio) were pre-exposed to 0.27mM ammonia (∼10% 96h LC50) for 3, 7 and 14days. Thereafter, each of these pre-exposed and parallel naïve groups were exposed to 1.35mM high environmental ammonia (HEA, ∼50% 96h LC50) for 12h and 48h to assess the occurrence of ammonia acclimation based on sub-lethal end-points, and to lethal ammonia concentrations (2.7mM, 96h LC50) in order to assess improved survival time. Results show that fish pre-exposed to ammonia for 3 and 7days had a longer survival time than the ammonia naïve fish. However, this effect disappeared after prolonged (14days) pre-exposure. Ammonia excretion rate (Jamm) was strongly inhibited (or even reversed) in the unacclimated groups during HEA. On the contrary, after 3days the pre-exposure fish maintained Jamm while after 7days these pre-acclimated fish were able to increase Jamm efficiently. Again, this effect disappeared after 14days of pre-acclimation. The efficient ammonia efflux in pre-acclimated fish was associated with the up-regulation of branchial mRNA expression of ammonia transporters and exchangers. Pre-exposure with ammonia for 3–7days stimulated an increment in the transcript level of gill Rhcg-a and Rhcg-b mRNA relative to the naïve control group and the up-regulation of these two Rhcg homologs was reinforced during subsequent HEA exposure. No effect of pre-exposure was noted for Rhbg. Relative to unacclimated fish, the transcript level of Na+/H+ exchangers (NHE-3) was raised in 3–7days pre-acclimated fish and remained higher during the subsequent HEA exposure while gill H+-ATPase activities and mRNA levels were not affected by pre-acclimation episodes. Likewise, ammonia pre-acclimated fish with or without HEA exposure displayed pronounced up-regulation in Na+/K+-ATPase activity and mRNA expression relative to the corresponding ammonia naïve groups. Overall, these data suggest that ammonia acclimation was evident for both lethal and the sub-lethal endpoints through priming mechanisms in ammonia excretory transcriptional processes, but these acclimation effects were transient and disappeared after prolonged pre-exposure.

Potential for Reducing On-Farm Greenhouse Gas and Ammonia Emissions from Dairy Cows with Prolonged Dietary Tannin Additions

Dairy cows are responsible for significant emissions of enteric methane (CH4) and produce nitrous oxide (N2O) and ammonia (NH3) gas from manure. As an abatement strategy, we explored the effects of long-term condensed tannin (Quebracho and chestnut extracts) addition to dairy cow diets. Previous studies have demonstrated that tannins in cow diets reduce methane and ammonia efflux, but none have done so over a >1-month time period. A modified stanchion barn equipped with gas analysis instrumentation measured CH4, N2O, and NH3 fluxes into and from the barn, at the onset of the experiment, and 45 and 90 days after feeding groups of lactating dairy cows a control diet or two levels of tannin extract at 0.45 and 1.8 % of dietary dry matter. Few statistical differences among treatments were observed, likely a consequence of high variability and low sample size necessary for conducting a study of this duration. However, on a per-cow basis, low and high tannin diets lowered CH4 emissions by 56 g cow−1 day−1 and by 48 g cow day−1, respectively. Diet tannin additions lowered CH4 (33 %), NH3 (23 %), and N2O (70 %) per unit milk corrected emissions in the high tannin treatment compared to the control at the end of the experiment, without significant loss in milk production. These results suggest that relatively low concentrations of diet tannin additions can reduce ruminant CH4 and gaseous N emissions from manure. The tannin effect observed after 90 days is a starting point for considering tannin additions as a potential long-term strategy for improving the environmental footprint of milk production.

An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito Aedes aegypti.

The transcripts of three putative ammonia (NH3/NH4 (+)) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na(+)/K(+)-ATPase. Ammonium concentration gradients were measured adjacent to anal papillae using the scanning ion-selective electrode technique (SIET) and used to calculate ammonia efflux by the anal papillae. dsRNA-mediated reductions in AeAmt1 decreased ammonia efflux at larval anal papillae and significantly increased ammonia levels in hemolymph, indicating a principal role for AeAmt1 in ammonia excretion. Pharmacological characterization of ammonia transport mechanisms in the anal papillae suggests that, in addition to AeAmt1, the ionomotive pumps V-type H(+)-ATPase and Na(+)/K(+)-ATPase as well as NHE3 are involved in ammonia excretion at the anal papillae.

Differential modulation of ammonia excretion, Rhesus glycoproteins and ion-regulation in common carp (Cyprinus carpio) following individual and combined exposure to waterborne copper and ammonia

The main objective of this study was to understand the mode of interaction between waterborne copper (Cu) and high environmental ammonia (HEA) exposure on freshwater fish, and how they influence the toxicity of each other when present together. For this purpose, individual and combined effects of Cu and HEA were examined on selected physiological and ion-regulatory processes and changes at transcript level in the common carp (Cyprinus carpio). Juvenile carp were exposed to 2.6μM Cu (25% of the 96h LC50value) and to 0.65mM ammonia (25% of the 96h LC50value) singly and as a mixture for 12h, 24h, 48h, 84h and 180h. Responses such as ammonia (Jamm) and urea (Jurea) excretion rate, plasma ammonia and urea, plasma ions (Na+, Cl− and K+), muscle water content (MWC) as well as branchial Na+/K+-ATPase (NKA) and H+-ATPase activity, and branchial mRNA expression of NKA, H+-ATPase, Na+/H+ exchanger (NHE-3) and Rhesus (Rh) glycoproteins were investigated under experimental conditions. Results show that Jamm was inhibited during Cu exposure, while HEA exposed fish were able to increase excretion efficiently. In the combined exposure, Jamm remained at the control levels indicating that Cu and HEA abolished each other’s effect. Expression of Rhcg (Rhcg-a and Rhcg-b) mRNA was upregulated during HEA, thereby facilitated ammonia efflux out of gills. On the contrary, Rhcg-a transcript level declined following Cu exposure which might account for Cu induced Jamm inhibition. Likewise, Rhcg-a was also down-regulated in Cu-HEA co-exposed fish whilst a temporary increment was noted for Rhch-b. Fish exposed to HEA displayed pronounced up-regulation in NKA expression and activity and stable plasma ion levels. In both the Cu exposure alone and combined Cu-HEA exposure, ion-osmo homeostasis was adversely affected, exemplified by the significant reduction in plasma [Na+] and [Cl−], and elevated plasma [K+], along with an elevation in MWC. These changes were accompanied by a decline in NKA activity. Gill H+-ATPase mRNA levels and activities were not affected by either Cu or HEA or both. Likewise, NHE-3 expression remained unaltered but tended to be numerically higher during HEA exposure. Overall, these data suggest that at equitoxic concentrations (25% of 96h LC50), the individual effect of Cu is more harmful while HEA induces quicker adaptive responses. Our findings also denote a competitive mode of interaction, exemplified by the inhibition of HEA -mediated adaptive responses in the presence of Cu.

Gut transport characteristics in herbivorous and carnivorous serrasalmid fish from ion-poor Rio Negro water.

Three closely related characids, Tambaqui (omnivore), black Piranha (carnivore), and Pacu (herbivore), all Serrasalmidae, inhabit the ion-poor, acidic Rio Negro. We compared O2-consumption and N excretion rates in vivo, and sodium, chloride, glucose, and ammonia transport characteristics of gut sac preparations in vitro. The Pacu had a significantly higher weight-specific oxygen consumption, and a lower N/Q ratio than the omnivorous Tambaqui, and a significantly lower urea-N excretion rate than the carnivorous black Piranha, suggesting N-limitation in the herbivorous Pacu. With a value of 2.62±0.15, gut to fork length ratio in the Pacu was about 2.5 times higher than in the black Piranha, and 2.0 times higher than in the Tambaqui. Anterior intestinal activities of three enzymes involved in N-fixation for amino acid synthesis (glutamate dehydrogenase, glutamate-oxaloacetate transferase, and glutamate-pyruvate transferase) were generally greatest in the carnivore and lowest in the herbivore species. In all three species, sodium, chloride, glucose, and ammonia were taken up at high rates from the intestine, resulting in an isosmotic fluid flux. Comparing the area-specific fluid flux of the anterior, mid, and posterior gut sections, no difference was detected between the three sections of the Pacu, while in the Tambaqui, it was highest in the anterior section, and in the black Piranha highest in the middle section. Overall, the area-specific uptake rates for sodium, chloride, glucose, and ammonia of anterior, mid, and posterior sections were similar in all three species, indicating that there is no difference in the area-specific transport rates associated with trophic position. The net ammonia uptake flux from gut interior was not significantly different from the net ammonia efflux to the serosal fluid, so that the ammonia removed from the intestine by the mucosal epithelium was quantitatively transferred through the tissue to the serosal side in all three species. Thus, metabolic activity of gut tissue did not significantly influence the net ammonia transfer. Due to the much higher gut to fork length ratio, the overall transport capacity of the gut of the herbivorous Pacu by far exceeded the transport capacity of their carnivorous and omnivorous relatives, thus compensating for the lower digestibility and the low Na(+), Cl(-), and N-content of the plant diet. Accordingly, in order to cope with the more difficult digestible plant material and the very low nitrogen content of plants, herbivorous fish have not evolved more effective area-specific transport capacities, but rather have increased the length of the gut.

Niche Differentiation of Ammonia-Oxidising Archaea (AOA) and Bacteria (AOB) in Response to Paper and Pulp Mill Effluent

Sediment organic loading has been shown to affect estuarine nitrification and denitrification, resulting in changes to sediment biogeochemistry and nutrient fluxes detrimental to estuarine health. This study examined the effects of organic loading on nutrient fluxes and microbial communities in sediments receiving effluent from a paper and pulp mill (PPM) by applying microcosm studies and molecular microbial ecology techniques. Three sites near the PPM outfall were compared to three control sites, one upstream and two downstream of the outfall. The control sites showed coupled nitrification-denitrification with minimal ammonia release from the sediment. In contrast, the impacted sites were characterised by nitrate uptake and substantial ammonia efflux from the sediments, consistent with a decoupling of nitrification and denitrification. Analysis of gene diversity demonstrated that the composition of nitrifier communities was not significantly different at the impacted sites compared to the control sites; however, analysis of gene abundance indicated that whilst there was no difference in total bacteria, total archaea or ammonia-oxidising archaea (AOA) abundance between the control and impacted sites, there was a significant reduction in ammonia-oxidising bacteria (AOB) at the impacted sites. The results of this study demonstrate an effect of organic loading on estuarine sediment biogeochemistry and highlight an apparent niche differentiation between AOA and AOB.

Cutaneous nitrogen excretion in the African clawed frog Xenopus laevis: Effects of high environmental ammonia (HEA)

Ammonia is a highly toxic molecule and often introduced in considerable amounts into aquatic environments due to anthropogenic activities. Many aquatic and semi-aquatic amphibians utilize, in addition to their kidneys, the skin for osmoregulation and nitrogen excretion. In the present study the effects of prolonged (7–21 days) exposure to high environmental ammonia (HEA, 1mmoll−1 NH4Cl) on cutaneous nitrogen excretion and gene expression of key-transporters involved in nitrogen excretion and acid-base regulation were investigated in the fully aquatic African clawed frog, Xenopus laevis. The study revealed that X. laevis excretes predominately ammonia of which approximately 50% is excreted via the skin. Both the ventral and dorsal skin were capable to generate a net ammonia efflux, which was significantly activated by 10mmoll−1 of the phosphodiesterase blocker theophylline. The obtained data further suggest that the ammonia efflux was promoted by an acidification of the unstirred boundary layer, likely generated by an apical localized V-ATPase, with NH3 being transported via cutaneous expressed ammonia transporters, Rhbg and Rhcg. Prolonged HEA exposure did significantly reduce the net-flux rates over the ventral skin with Vmax changing from 256nmolcm−2h−1 in control frogs to 196nmolcm−2h−1 in HEA exposed animals. Further, prolonged HEA exposure caused a decrease in mRNA expression levels of the ammonia transporter Rhbg, Na+/K+-ATPase (α-subunit) and V-ATPase (subunit H) in the ventral and dorsal skin and the kidney. In contrast, Rhcg expression levels were unaffected by HEA in skin tissues.

Benthic flux of oxygen and nutrients across Oregon/California shelf sediments

A free vehicle benthic chamber has been used to define benthic nutrient fluxes on the Southern Oregon and Northern California shelf (90–200m). We measured fluxes on two occasions, in the spring and fall of 2007, to assess variability in fluxes and bioirrigation rates. Chambers were also employed to assess the impact of changing oxygen content on benthic fluxes. Oxygen uptake rates are similar to within 30% between sites in Oregon, Northern California and Central California, suggesting that a large portion of the Western US continental shelf experiences the same oxygen demand. Oxygen uptake (−2 to −10mmolm−2d−1) at S. Oregon/N. California sites is likely responsible for <50% of the organic carbon (Corg) oxidized in these sediments as denitrification and other electron transport processes generate much of the observed TCO2 flux (4 to 31mmolCm−2d−1). Much like other coastal regions, these sediments are generally a net sink of water column fixed N as nitrate uptake rates (−0.6 to −2.2mmolNm−2d−1) typically exceed ammonium effluxes (0 to 1.9mmolNm−2d−1). As chamber oxygen concentrations decline during incubation, the concentration vs. time trends in phosphate, nitrate, and silicate remain linear. We interpret this to signify that there is no short-term change in the flux of these species as a response to changing oxygen concentration. However, there is a non-linear response for ammonium, with some ammonium fluxes increasing non-linearly by as much as a factor of 14. This occurs after an incubation period of 5–15h when oxygen levels are 50%–20% of their ambient values. This change in flux may be related to rather small changes in chamber oxygen concentration, suggesting that under hypoxic conditions, there could be an enhanced efflux of ammonia to the water column. This would provide a positive feedback leading to enhanced primary productivity and increasing hypoxia.

Sensitivity of ventilation and brain metabolism to ammonia exposure in rainbow trout,Oncorhynchus mykiss

Ammonia has been documented as a respiratory gas that stimulates ventilation, and is sensed by peripheral neuroepithelial cells (NECs) in the gills in ammoniotelic rainbow trout. However, the hyperventilatory response is abolished in trout chronically exposed (1+ months) to high environmental ammonia [HEA; 250 μmol l(-1) (NH4)2SO4]. This study investigates whether the brain is involved in the acute sensitivity of ventilation to ammonia, and whether changes in brain metabolism are related to the loss of hyperventilatory responses in trout chronically exposed to HEA ('HEA trout'). Hyperventilation (via increased ventilatory amplitude rather than rate) and increased total ammonia concentration ([TAmm]) in brain tissue were induced in parallel by acute HEA exposure in control trout in a concentration-series experiment [500, 750 and 1000 μmol l(-1) (NH4)2SO4], but these inductions were abolished in HEA trout. Ventilation was correlated more closely to [TAmm] in brain rather than to [TAmm] in plasma or cerebrospinal fluid. The close correlation of hyperventilation and increased brain [TAmm] also occurred in control trout acutely exposed to HEA in a time-series analysis [500 μmol l(-1) (NH4)2SO4; 15, 30, 45 and 60 min], as well as in a methionine sulfoxamine (MSOX) pre-injection experiment [to inhibit glutamine synthetase (GSase)]. These correlations consistently suggest that brain [TAmm] is involved in the hyperventilatory responses to ammonia in trout. The MSOX treatments, together with measurements of GSase activity, TAmm, glutamine and glutamate concentrations in brain tissue, were conducted in both the control and HEA trout. These experiments revealed that GSase plays an important role in transferring ammonia to glutamate to make glutamine in trout brain, thereby attenuating the elevation of brain [TAmm] following HEA exposure, and that glutamate concentration is reduced in HEA trout. The mRNAs for the ammonia channel proteins Rhbg, Rhcg1 and Rhcg2 were expressed in trout brain, and the expression of Rhbg and Rhcg2 increased in HEA trout, potentially as a mechanism to facilitate the efflux of ammonia. In summary, the brain appears to be involved in the sensitivity of ventilation to ammonia, and brain ammonia levels are regulated metabolically in trout.

Ammonia permeability of the soybean nodulin 26 channel

Soybean nodulin 26 (nod26), a member of the aquaporin superfamily, is the major protein component of the symbiosome membrane that encloses nitrogen-fixing bacteroids in root nodules. Previous work has demonstrated that nod26 facilitates the transport of water and glycerol, although a potential additional role as a channel for fixed ammonia efflux has been hypothesized. In the present study it is shown that recombinant nod26 reconstituted into proteoliposomes facilitates NH 3 transport in an Hg 2+-sensitive manner with a reduced activation energy, hallmarks of protein-facilitated transport characteristic of aquaporins. Comparison of the predicted single-channel transport rates of nod26 suggests a 4.9-fold preference for ammonia compared to water.

NHE4 is critical for the renal handling of ammonia in rodents

Ammonia absorption by the medullary thick ascending limb of Henle's loop (MTALH) is thought to be a critical step in renal ammonia handling and excretion in urine, in which it is the main acid component. Basolateral Na+/H+ exchangers have been proposed to play a role in ammonia efflux out of MTALH cells, which express 2 exchanger isoforms: Na+/H+ exchanger 1 (NHE1) and NHE4. Here, we investigated the role of NHE4 in urinary acid excretion and found that NHE4-/- mice exhibited compensated hyperchloremic metabolic acidosis, together with inappropriate urinary net acid excretion. When challenged with a 7-day HCl load, NHE4-/- mice were unable to increase their urinary ammonium and net acid excretion and displayed reduced ammonium medulla content compared with wild-type littermates. Both pharmacologic inhibition and genetic disruption of NHE4 caused a marked decrease in ammonia absorption by the MTALH. Finally, dietary induction of metabolic acidosis increased NHE4 mRNA expression in mouse MTALH cells and enhanced renal NHE4 activity in rats, as measured by in vitro microperfusion of MTALH. We therefore conclude that ammonia absorption by the MTALH requires the presence of NHE4 and that lack of NHE4 reduces the ability of MTALH epithelial cells to create the cortico-papillary gradient of NH3/NH4+ needed to excrete an acid load, contributing to systemic metabolic acidosis.

Functional characterization of Rhesus glycoproteins from an ammoniotelic teleost, the rainbow trout, using oocyte expression and SIET analysis

Recent experimental evidence from rainbow trout suggests that gill ammonia transport may be mediated in part via Rhesus (Rh) glycoproteins. In this study we analyzed the transport properties of trout Rh proteins (Rhag, Rhbg1, Rhbg2, Rhcg1, Rhcg2, Rh30-like) expressed in Xenopus oocytes, using the radiolabeled ammonia analogue [(14)C]methylamine, and the scanning ion electrode technique (SIET). All of the trout Rh proteins, except Rh30-like, facilitated methylamine uptake. Uptake was saturable, with K(m) values ranging from 4.6 to 8.9 mmol l(-1). Raising external pH from 7.5 to 8.5 resulted in 3- to 4-fold elevations in J(max) values for methylamine; K(m) values were unchanged when expressed as total or protonated methylamine. Efflux of methylamine was also facilitated in Rh-expressing oocytes. Efflux and influx rates were stimulated by a pH gradient, with higher rates observed with steeper H(+) gradients. NH(4)Cl inhibited methylamine uptake in oocytes expressing Rhbg1 or Rhcg2. When external pH was elevated from 7.5 to 8.5, the K(i) for ammonia against methylamine transport was 35-40% lower when expressed as total ammonia or NH(4)(+), but 5- to 6-fold higher when expressed as NH(3). With SIET we confirmed that ammonia uptake was facilitated by Rhag and Rhcg2, but not Rh30-like proteins. Ammonia uptake was saturable, with a comparable J(max) but lower K(m) value than for total or protonated methylamine. At low substrate concentrations, the ammonia uptake rate was greater than that of methylamine. The K(m) for total ammonia (560 micromol l(-1)) lies within the physiological range for trout. The results are consistent with a model whereby NH(4)(+) initially binds, but NH(3) passes through the Rh channels. We propose that Rh glycoproteins in the trout gill are low affinity, high capacity ammonia transporters that exploit the favorable pH gradient formed by the acidified gill boundary layer in order to facilitate rapid ammonia efflux when plasma ammonia concentrations are elevated.

A new paradigm for ammonia excretion in aquatic animals: role of Rhesus(Rh) glycoproteins

Ammonia excretion at the gills of fish has been studied for 80 years, but the mechanism(s) involved remain controversial. The relatively recent discovery of the ammonia-transporting function of the Rhesus (Rh) proteins, a family related to the Mep/Amt family of methyl ammonia and ammonia transporters in bacteria, yeast and plants, and the occurrence of these genes and glycosylated proteins in fish gills has opened a new paradigm. We provide background on the evolution and function of the Rh proteins, and review recent studies employing molecular physiology which demonstrate their important contribution to branchial ammonia efflux. Rhag occurs in red blood cells, whereas several isoforms of both Rhbg and Rhcg occur in many tissues. In the branchial epithelium, Rhcg appears to be localized in apical membranes and Rhbg in basolateral membranes. Their gene expression is upregulated during exposure to high environmental ammonia or internal ammonia infusion, and may be sensitive to synergistic stimulation by ammonia and cortisol. Rhcg in particular appears to be coupled to H(+) excretion and Na(+) uptake mechanisms. We propose a new model for ammonia excretion in freshwater fish and its variable linkage to Na(+) uptake and acid excretion. In this model, Rhag facilitates NH(3) flux out of the erythrocyte, Rhbg moves it across the basolateral membrane of the branchial ionocyte, and an apical "Na(+)/NH (+)(4) exchange complex" consisting of several membrane transporters (Rhcg, V-type H(+)-ATPase, Na(+)/H(+) exchanger NHE-2 and/or NHE-3, Na(+) channel) working together as a metabolon provides an acid trapping mechanism for apical excretion. Intracellular carbonic anhydrase (CA-2) and basolateral Na(+)/HCO (-)(3) cotransporter (NBC-1) and Na(+)/K(+)-ATPase play indirect roles. These mechanisms are normally superimposed on a substantial outward movement of NH(3) by simple diffusion, which is probably dependent on acid trapping in boundary layer water by H(+) ions created by the catalysed or non-catalysed hydration of expired metabolic CO(2). Profitable areas for future investigation of Rh proteins in fish are highlighted: their involvement in the mechanism of ammonia excretion across the gills in seawater fish, their possible importance in ammonia excretion across the skin, their potential dual role as CO(2) transporters, their responses to feeding, and their roles in early life stages prior to the full development of gills.

Nitrogen cycling in a deep ocean margin sediment (Sagami Bay, Japan)

On the basis of in situ NO3−1 microprofiles and chamber incubations complemented by laboratory‐based assessments of anammox and denitrification we evaluate the nitrogen turnover of an ocean margin sediment at 1450‐m water depth. In situ NO3−1 profiles horizontally separated by 12 mm reflected highly variable NO3−1 penetration depths, NO3−1 consumption rates, and nitrification. On average the turnover time of the pore‐water NO3−1 pool was ~0.2 d. Net release of NH4+ during mineralization (0.95 mmol m−2 d−1) sustained a net efflux of ammonia (53%), nitrification (24%), and anammox activity (23%). The sediment had a relatively high in situ net influx of NO3−1 (1.44 mmol m−2 d−1) that balanced the N2 production as assessed by onboard tracer experiments. N2 production was attributed to prokaryotic denitrification (59%), anammox (37%), and foraminifera‐based denitrification (4%). Anammox thereby represented an important nutrient sink, but the N2 production was dominated by denitrification. Despite the fact that NO3−1 stored inside foraminifera represented ~80% of the total benthic NO3−1 pool, the slow intracellular NO3−1 turnover that, on average, sustained foraminifera metabolism for 12‐52 d, contributed only to a minor extent to the overall N2 production. The microbial activity in the surface sediment is a net nutrient sink of ~1.1 mmol N m−2 d−1, which aligns with many studies performed in coastal and shelf environments. Continental margin areas can act as significant N sinks and play an important role in regional N budgets.

Ammonia transport in cultured gill epithelium of freshwater rainbow trout: the importance of Rhesus glycoproteins and the presence of an apical Na+/NH4+ exchange complex

The mechanisms of ammonia excretion at fish gills have been studied for decades but details remain unclear, with continuing debate on the relative importance of non-ionic NH(3) or ionic NH(4)(+) permeation by various mechanisms. The presence of an apical Na(+)/NH(4)(+) exchanger has also been controversial. The present study utilized an in vitro cultured gill epithelium (double seeded insert, DSI) of freshwater rainbow trout as a model to investigate these issues. The relationship between basolateral ammonia concentration and efflux to apical freshwater was curvilinear, indicative of a saturable carrier-mediated component (K(m)=66 micromol l(-1)) superimposed on a large diffusive linear component. Pre-exposure to elevated ammonia (2000 micromol l(-1)) and cortisol (1000 ng ml(-1)) had synergistic effects on the ammonia permeability of DSI, with significantly increased Na(+) influx and positive correlations between ammonia efflux and Na(+) uptake. This increase in ammonia permeability was bidirectional. It could not be explained by changes in paracellular permeability as measured by [(3)H]PEG-4000 flux. The mRNA expressions of Rhbg, Rhcg2, H(+)-ATPase and Na(+)/H(+) exchanger-2 (NHE-2) were up-regulated in DSI pre-exposed to ammonia and cortisol, CA-2 mRNA was down-regulated, and transepithelial potential became more negative. Bafilomycin (1 micromol l(-1)), phenamil (10 micromol l(-1)) and 5-(N,N-hexamethylene)amiloride (HMA, 10 micromol l(-1)) applied to the apical solution significantly inhibited ammonia efflux, indicating that H(+)-ATPase, Na(+) channel and NHE-2 pathways on the apical surface were involved in ammonia excretion. Apical amiloride (100 micromol l(-1)) was similarly effective, while basolateral HMA was ineffective. Pre-treatment with apical freshwater low in [Na(+)] caused increases in both Rhcg2 mRNA expression and ammonia efflux without change in paracellular permeability. These data suggest that Rhesus glycoproteins are important for ammonia transport in the freshwater trout gill, and may help to explain in vivo data where plasma ammonia stabilized at 50% below water levels during exposure to high environmental ammonia ( approximately 2300 micromol l(-1)). We propose an apical ;Na(+)/NH(4)(+) exchange complex' consisting of several membrane transporters, while affirming the importance of non-ionic NH(3) diffusion in ammonia excretion across freshwater fish gills.

Nitrogen budget for a low-salinity, zero-water exchange culture system: II. Evaluation of isonitrogenous feeding of various dietary protein levels to Litopenaeus vannamei (Boone)

This study evaluated the effects of isonitrogenous feeding (60 g dietary protein per kilogram of body weight per day) using experimental feeds with 25%, 30%, 35% and 40% protein on the nitrogen budget, ammonia efflux rate, growth and survival of juvenile Litopenaeus vannamei raised in a low-salinity (4 g L−1) zero-water exchange culture system for 4 weeks. No significant differences in weight gain or instantaneous growth rate were observed between the dietary treatments with 35% and 40% protein after 3 weeks of study, or between treatments with 25% and 30% protein after 4 weeks of study. High mortality rates were observed for the 35% and 40% protein treatments, probably associated with high nitrite levels (4.80 and 7.36 mg NO2-NL−1 respectively) in water. Among the various dietary treatments, 39–46.3% of feed nitrogen was converted to shrimp biomass, 32.8–38.0% and 14.4–39.9% remained within the system as organic and inorganic nitrogen, respectively, and 32.5–39.3% was unaccounted for. The results of the present study showed high nitrogen utilization efficiencies. However, as the nitrogen loading of the zero-water exchange system increased, so did the nitrogen excretion of shrimp, causing a deteriorated general condition of the shrimp, demonstrated by the low ammonia efflux rates recorded at the end of the trial. This study confirms that low-salinity closed systems are particularly susceptible to nitrogen loading. Thus, in these culture systems, low-protein feeds may perform better as they provide more carbon for heterotrophic bacteria and less nitrogen to be degraded and transformed into nitrogenous wastes.