Recently, a "Na(+)/NH(4)(+) 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 CO(2) 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 NH(4)HCO(3) 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.

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 þ uptake (J Na in ) in accordance with the current model for Na þ =NH 4 þ exchange in freshwater.Using divided chambers, branchial and cutaneous J amm , J Na in and oxygen consumption (MO 2 ) by larval rainbow trout were assessed. Following hatch, the skin accounted for 97% and 86% of total J amm and J Na in , respectively. J amm and J Na in shifted to the gills simultaneously at 15 days post-hatch (dph) and were highly correlated (R 2 ¼ 0.951) at the gills, but not the skin, over development. Contrastingly, MO 2 shifted significantly later at 27 dph, in agreement with the ionoregulatory hypothesis. Moreover, the mRNA expression and/ or enzymatic activity of Rhesus proteins, Naþ -ATPase and carbonic anhydrase, all key components of the Na þ =NH 4 þ -exchange system, increased in the gills over larval development.We propose that the ontogeny of branchial J Na in occurs as Na þ =NH 4 þ exchange and provide evidence for a novel element to the ionoregulatory hypothesis, the excretion of potentially lethal metabolic ammonia.

Loss-of-function technologies, such as morpholino-and RNAimediated gene knockdown, and TALEN-and CRISPR/Cas9mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses).

In rainbow trout, the dominant site of Na + uptake (J Na,in ) and ammonia excretion (J amm ) 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, J Na,in and J amm occur via loose Na + /NH 4 + 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, J Na,in by the yolk sac skin of PH trout occurs independently of J amm . Following a 12 h exposure to high environmental ammonia (HEA; 0.5 mmol l −1 NH 4 HCO 3 ; 600 µmol l −1 Na + ; pH 8), J amm 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 J Na,in across the gills only, supporting our hypothesis. Moreover, gene expression of proteins involved in J Na,in [Na -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 J Na,in and J amm by the dominant exchange epithelium at two distinct stages of early development are fundamentally different.

Recent molecular evidence points towards a capacity for ammonia transport across the skin of adult rainbow trout. A series of in vivo and in vitro experiments were conducted to understand the role of cutaneous ammonia excretion (J amm) under control conditions and after 12-h pre-exposure to high environmental ammonia (HEA; 2 mmol/l NH4HCO3). Divided chamber experiments with bladder-catheterized, rectally ligated fish under light anesthesia were performed to separate cutaneous J amm from branchial, renal, and intestinal J amm. Under control conditions, cutaneous J amm accounted for 4.5 % of total J amm in vivo. In fish pre-exposed to HEA, plasma total ammonia concentration increased 20-fold to approximately 1,000 μmol/l, branchial J amm increased 1.5- to 2.7-fold, and urinary J amm increased about 7-fold. Urinary J amm still accounted for less than 2 % of total J amm. Cutaneous J amm increased 4-fold yet amounted to only 5.7 % of total J amm in these fish. Genes (Rhcg1, Rhcg2, Rhbg, NHE-2, v-type H(+)-ATPase) known to be involved in ammonia excretion at the gills of trout were all expressed at the mRNA level in the skin, but their expression did not increase with HEA pre-exposure. In vitro analyses using [(14)C] methylamine (MA), an ammonia analog which is transported by Rh proteins, demonstrated that MA permeability in isolated skin sections was higher in HEA pre-exposed fish than in control fish. The addition of basolateral ammonia (1,000 μmol/l) to this system abolished this increase in permeability, suggesting ammonia competition with MA for Rh-mediated transport across the skin of HEA pre-exposed trout; this did not occur in skin sections from control trout. Moreover, in vitro J amm by the skin of fish which had been pre-exposed to HEA was also higher than in control fish in the absence of basolateral ammonia, pointing towards a possible cutaneous ammonia loading in response to HEA. In vitro MA permeability was reduced upon the addition of amiloride (10(-4) mol/l), but not phenamil (10(-5) mol/l) suggesting a role for a Na/H-exchanger (NHE) in cutaneous ammonia transport, as has been previously described in the skin of larval fish. Overall, it appears that under control conditions and in response to HEA pre-exposure, the skin makes only a very minor contribution to total J amm, but the observed increases in cutaneous J amm in vivo and in cutaneous J amm and MA permeability in vitro demonstrate the capacity for ammonia transport in the skin of adult trout. It remains unclear if this capacity may become significant under certain environmental challenges or if it is merely a remnant of cutaneous transport capacity from early life stages in these fish.

Purely diffusive O2 transport typically is insufficient to sustain aerobic metabolism in most multicellular organisms. In animals that are small enough, however, a high surface-to-volume ratio may allow passive diffusion alone to supply sufficient O2 transfer. The purpose of this study was to explore the impacts of internal convection on respiratory gas transfer in a small complex organism, the larval zebrafish ( Danio rerio). Specifically, we tested the hypothesis that internal convection is required for the normal transfer of the respiratory gases O2 and CO2 and maintenance of resting aerobic metabolic rate in larvae at 4 days postfertilization (dpf). Morpholino knockdown of the vascular endothelial growth factor (VEGF) or cardiac troponin T (TNNT2) proteins allowed an examination of gas transfer in two independent models lacking internal convection. With the use of a scanning micro-optrode technique to measure regional epithelial O2 fluxes ( Jo2), it was demonstrated that larvae lacking convection exhibited reduced Jo2 in regions spanning the head to the trunk. Moreover, the acute loss of internal convection caused by heart stoppage resulted in reduced rates of cutaneous Jo2, an effect that was reversed upon the restoration of internal convection. With the use of whole body respirometry, it was shown that loss of internal convection was associated with reduced resting rates of O2 consumption and CO2 excretion in larvae at 4 dpf. The results of these experiments clearly demonstrate that internal convection is required to maintain resting rates of respiratory gas transfer in larval zebrafish.

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Nitrogen metabolism in fishes has been a focus of comparative physiologists for nearly a century. In this Review, we focus specifically on early life stages of fishes, which have received considerable attention in more recent work. Nitrogen metabolism and excretion in early life differs fundamentally from that of juvenile and adult fishes because of (1) the presence of a chorion capsule in embryos that imposes a limitation on effective ammonia excretion, (2) an amino acid-based metabolism that generates a substantial ammonia load, and (3) the lack of a functional gill, which is the primary site of nitrogen excretion in juvenile and adult fishes. Recent findings have shed considerable light on the mechanisms by which these constraints are overcome in early life. Perhaps most importantly, the discovery of Rhesus (Rh) glycoproteins as ammonia transporters and their expression in ion-transporting cells on the skin of larval fishes has transformed our understanding of ammonia excretion by fishes in general. The emergence of larval zebrafish as a model species, together with genetic knockdown techniques, has similarly advanced our understanding of ammonia and urea metabolism and excretion by larval fishes. It has also now been demonstrated that ammonia excretion is one of the primary functions of the developing gill in rainbow trout larvae, leading to new hypotheses regarding the physiological demands driving gill development in larval fishes. Here, we highlight and discuss the dramatic changes in nitrogen handling that occur over early life development in fishes.