The Rhesus glycoprotein Rhcgb is expendable for ammonia excretion and Na+ uptake in zebrafish (Danio rerio)

Injection of antisense oligonucleotide morpholinos to elicit selective gene knockdown of ammonia (Rhag, Rhbg, and Rhcg1) or urea transporters (UT) was used as a tool to assess the relative importance of each transporter to nitrogen excretion in developing zebrafish ( Danio rerio). Knockdown of UT caused urea excretion to decrease by ∼90%, whereas each of the Rh protein knockdowns resulted in an ∼50% reduction in ammonia excretion. Contrary to what has been hypothesized previously for adult fish, each of the Rh proteins appeared to have a similar effect on total ammonia excretion, and thus all are required to facilitate normal ammonia excretion in the zebrafish larva. As demonstrated in other teleosts, zebrafish embryos utilized urea to a much greater extent than adults and were effectively ureotelic until hatching. At that point, ammonia excretion rapidly increased and appeared to be triggered by a large increase in the mRNA expression of Rhag, Rhbg, and Rhcg1. Unlike the situation in the adult pufferfish ( 35 ), the various transporters are not specifically localized to the gills of the developing zebrafish, but each protein has a unique expression pattern along the skin, gills, and yolk sac. This disparate pattern of expression would appear to preclude interaction between the Rh proteins in zebrafish embryos. However, this may be a developmental feature of the delayed maturation of the gills, because as the embryos matured, expression of the transporters in and around the gills increased.

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Sublethal Effects of Propiconazole On The Metabolism of Lambari Deuterodon Iguape (Eigenmann 1907), A Native Species From Brazil

10.21203/rs.3.rs-160456/v1 ◽

2021 ◽

Author(s):

Marcelo Barbosa Henriques ◽

Karina Fernandes Oliveira Rezende ◽

Leonardo Castilho-Barros ◽

Edison Barbieri

Keyword(s):

Danio Rerio ◽

Native Species ◽

Statistical Difference ◽

Sublethal Effects ◽

Metabolic Response ◽

Ammonia Excretion ◽

Native Fish ◽

Aquatic Environments ◽

Metabolic Rates ◽

Pesticide Contamination

Abstract The objective of this study was to analyze the sublethal effects of propiconazole on Deuterodon iguape a native fish common in Brazil, which has potential for aquaculture and use as a bioindicator. The hypothesis was to test whether D. iguape has a metabolism similar to Danio rerio so that its use in bioassays may be validated. Lethal concentration (LC50) and metabolic rates were studied in fish exposed to propiconazole. There was an increase and decrease in the metabolic rate at 0.01 and 0.1 µg L-1, with a statistical difference from the control ratio for the two species. The same trend towards ammonia excretion was observed, initially increasing to a concentration of 0.01 µg L-1 and then decreasing as the concentration of propiconazole was increased. The averages of specific ammonia excretion in all concentrations were significantly different compared to the control. It was concluded that exposure to propiconazole increased the metabolic rates of D. Iguape and D. rerio, up to 0.05 µg L-1, and decreased the rates when the concentration was doubled (0.1 µg L-1); causing serious problems in fish metabolism. D. iguape proved to be a good and useful bioindicator for ichthyologists or ecologists in studies of moderate pesticide contamination in freshwater aquatic environments, as its metabolic response was similar to D. rerio.

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Use of a carbonic anhydrase Ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio)

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00215.2020 ◽

2021 ◽

Vol 320 (1) ◽

pp. R55-R68

Author(s):

Alex M. Zimmer ◽

Milica Mandic ◽

Hong Meng Yew ◽

Emma Kunert ◽

Yihang K. Pan ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Danio Rerio ◽

Ammonia Excretion ◽

Ion Uptake ◽

Whole Body ◽

Acid Base Balance ◽

Acid Base ◽

Uptake Mechanism ◽

Uptake Rates ◽

Base Balance

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish ( Danio rerio) is expressed abundantly in Na+-absorbing/H+-secreting H+-ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a−/− mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a−/− mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na+ uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na+ content remained constant. While Cl− uptake was significantly reduced in ca17a−/− mutants, Cl− content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl− uptake, implicating Ca17a in the mechanism of Cl− uptake by larval zebrafish. H+ secretion, O2 consumption, CO2 excretion, and ammonia excretion were generally unaltered in ca17a−/− mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl− uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.

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Sodium absorption coupled to ammonia excretion in osmoconforming marine invertebrates

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1986.251.5.r957 ◽

1986 ◽

Vol 251 (5) ◽

pp. R957-R962 ◽

Cited By ~ 2

Author(s):

K. C. Hunter ◽

L. B. Kirschner

Keyword(s):

Cation Exchange ◽

Marine Invertebrates ◽

Ammonia Excretion ◽

Mytilus Californianus ◽

Sodium Absorption ◽

Proton Efflux ◽

Petrolisthes Cinctipes ◽

Na Uptake

Evidence was sought for the presence of amiloride-sensitive Na-NH4 and NaH exchange systems in four species of marine osmoconformers. When the crabs Cancer antennarius and Petrolisthes cinctipes were in seawater (SW), amiloride (10(-4) M) reduced NH3 efflux by approximately 33 and 60%. Inhibition was reversible on removing the amiloride. In dilute (60%) SW, inhibition of NH3 output by C. antennarius was even greater than in SW. Na+ uptake by P. cinctipes was reduced by approximately 20% in the presence of amiloride (the measurement was not made on C. antennarius). Amiloride had no effect on proton efflux in either crab. The data suggest that Na-NH4 exchange occurs in these animals but that Na-H exchange does not occur. The inhibitor had no effect on NH+4 excretion by the polychaete worm Nephtys caecoides and the mussel Mytilus californianus; it was also without effect on proton output by the worm. The data suggest that the exchange systems are absent from these animals. Implications of these observations for the evolution of such cation exchange systems are discussed.

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Hydrogen sulfide inhibits Na+ uptake in larval zebrafish, Danio rerio

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-014-1550-y ◽

2014 ◽

Vol 467 (4) ◽

pp. 651-664 ◽

Cited By ~ 9

Author(s):

Yusuke Kumai ◽

Cosima S. Porteus ◽

Raymond W. M. Kwong ◽

Steve F. Perry

Keyword(s):

Hydrogen Sulfide ◽

Danio Rerio ◽

Larval Zebrafish ◽

Na Uptake

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Ammonia excretion via Rhcg1 facilitates Na+ uptake in larval zebrafish, Danio rerio, in acidic water

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00282.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. R1517-R1528 ◽

Cited By ~ 61

Author(s):

Yusuke Kumai ◽

Steve F. Perry

Keyword(s):

Freshwater Fish ◽

Ammonia Excretion ◽

Acidic Water ◽

Chemical Gradients ◽

Larval Zebrafish ◽

Zebrafish Larvae ◽

Pharmacological Data ◽

Water Ph ◽

Rh Proteins ◽

Na Uptake

The involvement of a Na+/H+ exchanger (NHE) in mediating Na+ uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na+ uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na+ uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-( N-ethyl- N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na+ uptake in fish reared in acidic water (pH ∼ 4.0). A role for NHE in Na+ uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na+/H+ exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na+ uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na+ uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na+ uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H+-ATPase) significantly reduced Na+ uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na+ uptake in larval zebrafish in acidic water: one in which Na+ uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H+-ATPase.

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