Physiological responses of European sea bass (Dicentrarchus labrax) exposed to increased carbon dioxide and reduced seawater salinities.

BACKGROUND: The iono- and osmoregulatory capacities of marine teleosts, such as European sea bass (Dicentrarchus labrax) are expected to be challenged by high carbon dioxide exposure, and the adverse effects of elevated CO2 could be amplified when such fish migrate into less buffered hypo-osmotic estuarine environments. Therefore, the effects of increased CO2 on the physiological responses of European sea bass (Dicentrarchus labrax) acclimated to 32 ppt, 10 ppt and 2.5 ppt were investigated. METHODS: Following acclimation to different salinities for two weeks, fish were exposed to present-day (400 µatm) and future (1000 µatm) atmospheric CO2 for 1, 3, 7 and 21 days. Blood pH, plasma ions (Na+, K+, Cl-), branchial mRNA expression of ion transporters such as Na+/K+-ATPase (NKA), Na+/K+/2Cl- co-transporters (NKCC) and ammonia transporters (e.g. Rhesus glycoproteins Rhbg, Rhcg1 and Rhcg2) were examined to understand the iono- and osmoregulatory consequences of elevated CO2. RESULTS: A transient but significant increase in the blood pH of exposed fish acclimated at 10 ppt (day 1) and 2.5 ppt (day 21) was observed possibly due to an overshoot of the blood HCO3- accumulation while a significant reduction of blood pH was observed after 21 days at 2.5ppt. However, no change was seen at 32 ppt. Generally, Na + concentration of control fish was relatively higher at 10 ppt and lower at 2.5 ppt compared to 32 ppt control group at all sampling periods. Additionally, NKA was upregulated in gill of juvenile sea bass when acclimated to lower salinities compared to 32 ppt control group. CO2 exposure generally downregulated NKA mRNA expression at 32ppt (day 1), 10 ppt (days 3, 7 and 21) and 2.5ppt (days 1 and 7) and also a significant reduction of NKCC mRNA level of the exposed fish acclimated at 32 ppt (1-3 days) and 10 ppt (7-21 days) was observed. Furthermore, Rhesus glycoproteins were generally upregulated in the fish acclimated at lower salinities indicating a higher dependance on gill ammonia excretion. Increased CO2 led to a reduced expression of Rhbg and may therefore reduce ammonia excretion rate. CONCLUSION: Juvenile sea bass were relatively successful in keeping acid base balance under an ocean acidification scenario. However, this came at a cost for ionoregulation with reduced NKA, NKCC and Rhbg expression rates as a consequence.

Ontogeny of urea and ammonia transporters in mahi-mahi (Coryphaena hippurus) early life stages.

The mechanism(s) of ammonia and urea excretion in freshwater fish have received considerable attention; however, parallel investigations of seawater fish, specifically in the early life stages are scarce. The first objective of this study was to evaluate the patterns of ammonia and urea excretion in mahi-mahi (Coryphaena hippurus) up to 102  hours post fertilization (hpf). Similar to other teleosts, mahi embryos are ureotelic before hatch and gradually switch to being ammoniotelic around the time of hatch. The second objective was to characterize mRNA levels of ammonia transporters (Rhag, Rhbg, Rhcg1 and Rhcg2), as well as urea transporter (UT) and sodium hydrogen exchangers (NHE3 and NHE2) during mahi development. As predicted, the mRNA levels of the Rhesus glycoprotein (Rh) genes, especially Rhag, Rhbg and the UT gene were highly consistent with the ontogeny of ammonia and urea excretion rates. Further, the localization of each transporter was examined in larvae collected at 60 and 102 hpf using in situ hybridization. Rhag was expressed in the gills, yolk sac, and operculum. Rhbg was expressed in the gills and upper mouth. Rhcg1 and NHE3 were co-localized in the sub-operculum, and Rhcg2 was expressed in the skin. Together, these results indicate that urea excretion is critical for ammonia detoxification during embryonic development and that Rh proteins are involved in ammonia excretion via gills and yolk sac, possibly facilitated by NHE3.

Evidence that Rh proteins in the anal papillae of the freshwater mosquito Aedes aegypti are involved in the regulation of acid-base balance in elevated salt and ammonia environments.

Aedes aegypti commonly inhabit ammonia-rich sewage effluents in tropical regions of the world where the adults are responsible for the spread of disease. Studies have shown the importance of the anal papillae of A. aegypti in ion uptake and ammonia excretion. The anal papillae express ammonia transporters and Rhesus (Rh) proteins which are involved in ammonia excretion and studies have primarily focused on understanding these mechanisms in freshwater. In this study, effects of rearing larvae in salt (5 mmol l-1 NaCl) or ammonia (5 mmol l-1 NH4Cl) on physiological endpoints of ammonia and ion regulation were assessed. In anal papillae of NaCl-reared larvae, Rh protein expression increased, NHE3 transcript abundance decreased and NH4+ excretion increased, and this coincided with decreased hemolymph [NH4+] and pH. We propose that under these conditions, larvae excrete more NH4+ through Rh proteins as a means of eliminating acid from the hemolymph. In anal papillae of NH4Cl-reared larvae, expression of an apical ammonia transporter and the Rh proteins decreased, the activities of NKA and VA decreased and increased, respectively, and this coincided with hemolymph acidification. The results present evidence for a role of Rh proteins in acid-base balance in response to elevated levels of salt, whereby ammonia is excreted as an acid equivalent.

Different mechanisms of Na+ uptake and ammonia excretion by the gill and yolk sac epithelium of early life stage rainbow trout.

In rainbow trout, the dominant site of Na+ uptake (JNa,in) and ammonia excretion (Jamm) shifts from the skin to the gills over development. Post-hatch (PH; 7 days post-hatch) larvae utilize the yolk sac skin for physiological exchange, whereas by complete yolk sac absorption (CYA; 30 days post-hatch), the gill is the dominant site. At the gills, JNa,in and Jamm occur via loose Na+/NH4+ exchange, but this exchange has not been examined in the skin of larval trout. Based on previous work, we hypothesized that, contrary to the gill model, JNa,in by the yolk sac skin of PH trout occurs independently of Jamm Following a 12 h exposure to high environmental ammonia (HEA; 0.5 mmol l-1 NH4HCO3; 600 µmol l-1 Na+; pH 8), Jamm by the gills of CYA trout and the yolk sac skin of PH larvae, which were isolated using divided chambers, increased significantly. However, this was coupled to an increase in JNa,in across the gills only, supporting our hypothesis. Moreover, gene expression of proteins involved in JNa,in [Na+/H+-exchanger-2 (NHE2) and H+-ATPase] increased in response to HEA only in the CYA gills. We further identified expression of the apical Rhesus (Rh) proteins Rhcg2 in putative pavement cells and Rhcg1 (co-localized with apical NHE2 and NHE3b and Na+/K+-ATPase) in putative peanut lectin agglutinin-positive (PNA+) ionocytes in gill sections. Similar Na+/K+-ATPase-positive cells expressing Rhcg1 and NHE3b, but not NHE2, were identified in the yolk sac epithelium. Overall, our findings suggest that the mechanisms of JNa,in and Jamm by the dominant exchange epithelium at two distinct stages of early development are fundamentally different.

Physiological and molecular responses of the goldfish (Carassius auratus) kidney to metabolic acidosis, and potential mechanisms of renal ammonia transport.

Relative to the gills, the mechanisms by which the kidney contributes to ammonia and acid-base homeostasis in fish are poorly understood. Goldfish were exposed to a low pH environment (pH 4.0, 48 h), which induced a characteristic metabolic acidosis and an increase in total plasma [ammonia] but reduced plasma ammonia partial pressure (PNH3). In the kidney tissue, total ammonia, lactate and intracellular pH remained unchanged. The urinary excretion rate of net base under control conditions changed to net acid excretion under low pH, with contributions from both the NH4 (+) (∼30%) and titratable acidity minus bicarbonate (∼70%; TA-HCO3 (-)) components. Inorganic phosphate (Pi), urea and Na(+) excretion rates were also elevated while Cl(-) excretion rates were unchanged. Renal alanine aminotransferase activity increased under acidosis. The increase in renal ammonia excretion was due to significant increases in both the glomerular filtration and the tubular secretion rates of ammonia, with the latter accounting for ∼75% of the increase. There was also a 3.5-fold increase in the mRNA expression of renal Rhcg-b (Rhcg1) mRNA. There was no relationship between ammonia secretion and Na(+) reabsorption. These data indicate that increased renal ammonia secretion during acidosis is probably mediated through Rhesus (Rh) glycoproteins and occurs independently of Na(+) transport, in contrast to branchial and epidermal models of Na(+)-dependent ammonia transport in freshwater fish. Rather, we propose a model of parallel H(+)/NH3 transport as the primary mechanism of renal tubular ammonia secretion that is dependent on renal amino acid catabolism.

Rh protein expression in branchial neuroepithelial cells, and the role of ammonia in ventilatory control in fish.

Bill Milsom has made seminal contributions to our understanding of ventilatory control in a wide range of vertebrates. Teleosts are particularly interesting, because they produce a 3rd, potentially toxic respiratory gas (ammonia) in large amounts. Fish are well known to hyperventilate under high environmental ammonia (HEA), but only recently has the potential role of ammonia in normal ventilatory control been investigated. It is now clear that ammonia can act directly as a ventilatory stimulant in trout, independent of its effects on acid-base balance. Even in ureotelic dogfish sharks, acute elevations in ammonia cause increases in ventilation. Peripherally, the detection of elevated ammonia resides in gill arches I and II in trout, and in vitro, neuroepithelial cells (NECs) from these arches are sensitive to ammonia, responding with elevations in intracellular Ca(2+) ([Ca(2+)]i). Centrally, hyperventilatory responses to ammonia correlate more closely with concentrations of ammonia in the brain than in plasma or CSF. After chronic HEA exposure, ventilatory responsiveness to ammonia is lost, associated with both an attenuation of the [Ca(2+)]i response in NECs, and the absence of elevation in brain ammonia concentration. Chronic exposure to HEA also causes increases in the mRNA expression of several Rh proteins (ammonia-conductive channels) in both brain and gills. "Single cell" PCR techniques have been used to isolate the individual responses of NECs versus other gill cell types. We suggest several circumstances (post-feeding, post-exercise) where the role of ammonia as a ventilatory stimulant may have adaptive benefits for O2 uptake in fish.

An Update on Kidney Ammonium Transport Along the Nephron.

Acid-base homeostasis is critical to the maintenance of normal health. The kidneys have a central role in bicarbonate generation, which occurs through the process of net acid excretion. Renal ammonia excretion is the predominant component of renal net acid excretion under basal conditions and in response to acid-base disturbances. Ammonia produced in the kidney is selectively transported into the urine or the renal vein. The amount of ammonia produced by the kidney that is excreted in the urine varies dramatically in response to physiological stimuli. Recent studies have advanced our understanding of ammonia metabolism's molecular mechanisms and regulation. Ammonia transport has been advanced by recognizing that the specific transport of NH3 and NH4+ by specific membrane proteins is critical to ammonia transport. Other studies show that proximal tubule protein, NBCe1, specifically the A variant, significantly regulates renal ammonia metabolism. This review discusses these critical aspects of the emerging features of ammonia metabolism and transport.

The effects of Deepwater Horizon crude oil on ammonia and urea handling in mahi-mahi (Coryphaena hippurus) early life stages.

Many ecologically important fishes, including mahi-mahi (Coryphaena hippurus), and their offspring were directly exposed to crude oil following the Deepwater Horizon (DWH) oil spill. Early life stage fish are especially vulnerable to the toxicity of crude oil-derived polycyclic aromatic hydrocarbons (PAHs). In teleosts, yolk sac proteins are the main energy source during development and are usually catabolized into ammonia or urea among other byproducts. Although excretion of these waste products is sensitive to oil exposure, we know little about the underlying mechanisms of this process. In this study, we examined the effects of crude oil on ammonia and urea handling in the early life stages of mahi. Mahi embryos exposed to 30-32 µg L-1 ∑PAH exhibited increased urea excretion rates and greater accumulation of urea in the tissues before hatch suggesting that ammonia, which is highly toxic, was converted into less-toxic urea. Oil-exposed embryos (6.3-32 µg L-1 ∑PAH) displayed significantly increased tissue ammonia levels at 42 hpf and upregulated mRNA levels of ammonia transporters (Rhag, Rhbg and Rhcg1) from 30 to 54 hpf. However, despite increased accumulation and higher expression of ammonia transporters, the larvae exposed to higher ∑PAH (30 µg L-1 ∑PAH) showed reduced ammonia excretion rates after hatch. Together, the increased production of nitrogenous waste reinforces previous work that increased energy demand in oil-exposed embryos is fueled, at least in part, by protein metabolism and that urea synthesis plays a role in ammonia detoxification in oil-exposed mahi embryos. To our knowledge, this study is the first to combine physiological and molecular approaches to assess the impact of crude-oil on both nitrogenous waste excretion and accumulation in the early life stages of any teleosts.

Ammonia Excretion in an Osmoregulatory Syncytium Is Facilitated by AeAmt2, a Novel Ammonia Transporter in Aedes aegypti Larvae.

The larvae of the mosquito Aedes aegypti inhabit ammonia rich septic tanks in tropical regions of the world that make extensive use of these systems, explaining the prevalence of disease during dry seasons. Since ammonia (NH3/[Formula: see text]) is toxic to animals, an understanding of the physiological mechanisms of ammonia excretion permitting the survival of A. aegypti larvae in high ammonia environments is important. We have characterized a novel ammonia transporter, AeAmt2, belonging to the Amt/MEP/Rh family of ammonia transporters. Based on the amino acid sequence, the predicted topology of AeAmt2 consists of 11 transmembrane helices with an extracellular N-terminus and a cytoplasmic C-terminus region. Alignment of the predicted AeAmt2 amino acid sequence with other Amt/MEP proteins from plants, bacteria, and yeast highlights the presence of conserved residues characteristic of ammonia conducting channels in this protein. AeAmt2 is expressed in the ionoregulatory anal papillae of A. aegypti larvae where it is localized to the apical membrane of the epithelium. dsRNA-mediated knockdown of AeAmt2 results in a significant decrease in [Formula: see text] efflux from the anal papillae, suggesting a key role in facilitating ammonia excretion. The effect of high environmental ammonia (HEA) on expression of AeAmt2, along with previously characterized AeAmt1, AeRh50-1, and AeRh50-2 in the anal papillae was investigated. We show that changes in expression of ammonia transporters occur in response to acute and chronic exposure to HEA, which reflects the importance of these transporters in the physiology of life in high ammonia habitats.

(Uncommon) Mechanisms of Branchial Ammonia Excretion in the Common Carp (Cyprinus carpio) in Response to Environmentally Induced Metabolic Acidosis.

Freshwater fishes generally increase ammonia excretion in acidic waters. The new model of ammonia transport in freshwater fish involves an association between the Rhesus (Rh) protein Rhcg-b, the Na(+)/H(+) exchanger (NHE), and a suite of other membrane transporters. We tested the hypothesis that Rhcg-b and NHE3 together play a critical role in branchial ammonia excretion in common carp (Cyprinus carpio) chronically exposed to a low-pH environment. Carp were exposed to three sequential environmental treatments-control pH 7.6 water (24 h), pH 4.0 water (72 h), and recovery pH 7.6 water (24 h)-or in a separate series were simply exposed to either control (72 h) or pH 4.0 (72 h) water. Branchial ammonia excretion was increased by ∼2.5-fold in the acid compared with the control period, despite the absence of an increase in the plasma-to-water partial pressure NH3 gradient. Alanine aminotransferase activity was higher in the gills of fish exposed to pH 4 versus control water, suggesting that ammonia may be generated in gill tissue. Gill Rhcg-b and NHE3b messenger RNA levels were significantly elevated in acid-treated relative to control fish, but at the protein level Rhcg-b decreased (30%) and NHE3b increased (2-fold) in response to water of pH 4.0. Using immunofluorescence microscopy, NHE3b and Rhcg-b were found to be colocalized to ionocytes along the interlamellar space of the filament of control fish. After 72 h of acid exposure, Rhcg-b staining almost disappeared from this region, and NHE3b was more prominent along the lamellae. We propose that ammoniagenesis within the gill tissue itself is responsible for the higher rates of branchial ammonia excretion during chronic metabolic acidosis. Unexpectedly, gill Rhcg-b does not appear to be important in gill ammonia transport in low-pH water, but the strong induction of NHE3b suggests that some NH4(+) may be eliminated directly in exchange for Na(+). These findings contrast with previous studies in larval zebrafish (Danio rerio) and medaka (Oryzias latipes), underlining the importance of species comparisons.