scholarly journals Respirometry and cutaneous oxygen flux measurements reveal a negligible aerobic cost of ion regulation in larval zebrafish (Danio rerio)

2020 ◽  
Vol 223 (18) ◽  
pp. jeb226753
Author(s):  
Julian J. Parker ◽  
Alex M. Zimmer ◽  
Steve F. Perry
Keyword(s):  
Danio Rerio ◽  
Oxygen Flux ◽  
Whole Body ◽  
Uptake Capacity ◽  
Ion Regulation ◽  
Larval Zebrafish ◽  
Morpholino Knockdown ◽  
Rearing Conditions ◽  
Flux Measurements ◽  
Energy Consuming

ABSTRACTFishes living in fresh water counter the passive loss of salts by actively absorbing ions through specialized cells termed ionocytes. Ionocytes contain ATP-dependent transporters and are enriched with mitochondria; therefore ionic regulation is an energy-consuming process. The purpose of this study was to assess the aerobic costs of ion transport in larval zebrafish (Danio rerio). We hypothesized that changes in rates of Na+ uptake evoked by acidic or low Na+ rearing conditions would result in corresponding changes in whole-body oxygen consumption (ṀO2) and/or cutaneous oxygen flux (JO2), measured at the ionocyte-expressing yolk sac epithelium using the scanning micro-optrode technique (SMOT). Larvae at 4 days post-fertilization (dpf) that were reared under low pH (pH 4) conditions exhibited a higher rate of Na+ uptake compared with fish reared under control conditions (pH 7.6), yet they displayed a lower ṀO2 and no difference in cutaneous JO2. Despite a higher Na+ uptake capacity in larvae reared under low Na+ conditions, there were no differences in ṀO2 and JO2 at 4 dpf. Furthermore, although Na+ uptake was nearly abolished in 2 dpf larvae lacking ionocytes after morpholino knockdown of the ionocyte proliferation regulating transcription factor foxi3a, ṀO2 and JO2 were unaffected. Finally, laser ablation of ionocytes did not affect cutaneous JO2. Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na+, are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence that ion regulation in zebrafish larvae incurs a low aerobic cost.

Estrogen-related receptor γ2 controls NaCl uptake to maintain ionic homeostasis

2021 ◽  
Author(s):  
Shang-Wu Shih ◽  
Jia-Jiun Yan ◽  
Yi-Hsing Wang ◽  
Yi-Ling Tsou ◽  
Ling Chiu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Body Fluid ◽  
Whole Body ◽  
Ionic Homeostasis ◽  
Ion Regulation ◽  
Expression Levels ◽  
Morpholino Knockdown ◽  
Oryzias Melastigma ◽  
Euryhaline Teleost

Estrogen-related receptors (ERRs) are known to function in mammalian kidney as key regulators of ion transport-related genes; however, a comprehensive understanding of the physiological functions of ERRs in vertebrate body fluid ionic homeostasis is still elusive. Here, we used medaka (Oryzias melastigma), a euryhaline teleost, to investigate how ERRs are involved in ion regulation. After transferring medaka from hypertonic seawater to hypotonic freshwater (FW), the mRNA expression levels of errγ2 were highly upregulated, suggesting that ERRγ2 may play a crucial role in ion uptake. In situ hybridization and immunofluorescence staining showed that errγ2 was specifically expressed in ionocytes, the cells responsible for Na+/Cl- transport. In normal FW, ERRγ2 morpholino knockdown caused reductions in the mRNA expression of Na+/Cl- cotransporter (NCC), the number of NCC ionocytes, Na+/Cl- influxes of ionocytes, and whole-body Na+/Cl- contents. In FW with low Na+ and low Cl-, the expression levels of mRNA for Na+/H+ exchanger 3 (NHE3) and NCC were both decreased in ERRγ2 morphants. Treating embryos with DY131, an agonist of ERRγ, increased the whole-body Na+/Cl- contents and ncc mRNA expression in ERRγ2 morphants. As such, medaka ERRγ2 may control Na+/Cl- uptake by regulating ncc and/or nhe3 mRNA expression and ionocyte number, and these regulatory actions may be subtly adjusted depending on internal and external ion concentrations. These findings not only provide new insights into the underpinning mechanism of actions of ERRs, but also enhance our understanding of their roles in body fluid ionic homeostasis for adaptation to changing environments during vertebrate evolution.

scholarly journals Angiotensin-II promotes Na+ uptake in larval zebrafish, Danio rerio, in acidic and ion-poor water

2013 ◽  
Vol 220 (3) ◽  
pp. 195-205 ◽  
Author(s):  
Yusuke Kumai ◽  
Nicholas J Bernier ◽  
Steve F Perry
Keyword(s):  
Angiotensin Ii ◽  
Danio Rerio ◽  
Glucocorticoid Receptors ◽  
Enzyme Inhibitor ◽  
Renin Angiotensin System ◽  
Whole Body ◽  
Ang Ii ◽  
Larval Zebrafish ◽  
Rapid Activation ◽  
Stimulation Of

The contribution of the renin–angiotensin system (RAS) to Na+uptake was investigated in larval zebrafish (Danio rerio). At 4 days post fertilization (dpf), the level of whole-body angiotensin-II (ANG-II) was significantly increased after 1- or 3-h exposure to acidic (pH=4.0) or ion-poor water (20-fold dilution of Ottawa tapwater), suggesting rapid activation of the RAS. Long-term (24 h) treatment of 3 dpf larvae with ANG-I or ANG-II significantly increased Na+uptake which was accompanied by an increase in mRNA expression of the Na+-Cl−cotransporter (zslc12a10.2). Induction of Na+uptake by exposure to ANG-I was blocked by simultaneously treating larvae with lisinopril (an angiotensin-converting enzyme inhibitor). Acute (2 h) exposure to acidic water or ion-poor water led to significant increase in Na+uptake which was partially blocked by the ANG-II receptor antagonist, telmisartan. Consistent with these data, translational knockdown of renin prevented the stimulation of Na+uptake following exposure to acidic or ion-poor water. The lack of any effects of pharmacological inhibition (using RU486), or knockdown of glucocorticoid receptors on the stimulation of Na+uptake during acute exposure to acidic or ion-poor environments, indicates that the acute effects of RAS occur independently of cortisol signaling. The results of this study demonstrate that the RAS is involved in Na+homeostasis in larval zebrafish.

scholarly journals Involvement of the calcium-sensing receptor in calcium homeostasis in larval zebrafish exposed to low environmental calcium

2014 ◽  
Vol 306 (4) ◽  
pp. R211-R221 ◽  
Author(s):  
Raymond W. M. Kwong ◽  
Dan Auprix ◽  
Steve F. Perry
Keyword(s):  
In Situ Hybridization ◽  
Confocal Microscopy ◽  
Mrna Expression ◽  
Danio Rerio ◽  
Whole Body ◽  
Calcium Sensing Receptor ◽  
Larval Zebrafish ◽  
Calcium Sensing ◽  
Normal Water

The involvement of the calcium-sensing receptor (CaSR) in Ca2+ homeostasis was investigated in larval zebrafish, Danio rerio. The expression of CaSR mRNA was first observed at 3 h posfertilization (hpf) and increased with development until plateauing at ∼48 hpf. At 4 dpf, CaSR mRNA was increased in fish acclimated to low Ca2+ water (25 μM vs. 250 μM in normal water). Using immunohistochemistry and confocal microscopy, we demonstrated that the CaSR is expressed in the olfactory epithelium, neuromasts, ionocytes on the yolk sac epithelium, and corpuscles of Stannius. Results of double immunohistochemistry and/or in situ hybridization indicated that the CaSR is localized to a subset of mitochondrion-rich ionocytes enriched with Na+/K+-ATPase and epithelial Ca2+ channel ( ecac). Translational knockdown of the CaSR prevented 4 dpf larvae from regulating whole body Ca2+ levels when exposed to a low Ca2+ environment. Further, the increases in ecac mRNA expression and Ca2+ influx, normally associated with exposure to low-Ca2+ water, were prevented by CaSR knockdown. These findings demonstrate that larval zebrafish lacking the CaSR lose their ability to regulate Ca2+ when confronted with a low-Ca2+ environment. Results from real-time PCR suggested that the mRNA expression of the hypocalcemic hormone stanniocalcin ( stc-1) remained elevated in the CaSR morphants following acclimation to low-Ca2+ water. Overall, the results suggest that the CaSR is critical for Ca2+ homeostasis in larval zebrafish exposed to low environmental Ca2+ levels, possibly owing to its modulation of stanniocalcin mRNA expression.

scholarly journals The tight junction protein claudin-b regulates epithelial permeability and sodium handling in larval zebrafish, Danio rerio

2013 ◽  
Vol 304 (7) ◽  
pp. R504-R513 ◽  
Author(s):  
Raymond W. M. Kwong ◽  
Steve F. Perry
Keyword(s):  
Tight Junction ◽  
Danio Rerio ◽  
Free Water ◽  
Yolk Sac ◽  
Tight Junction Protein ◽  
Whole Body ◽  
Epithelial Permeability ◽  
Pericardial Cavity ◽  
Larval Zebrafish ◽  
Junction Protein

The functional role of the tight junction protein claudin-b in larval zebrafish ( Danio rerio) was investigated. We showed that claudin-b protein is expressed at epithelial cell-cell contacts on the skin. Translational gene knockdown of claudin-b protein expression caused developmental defects, including edema in the pericardial cavity and yolk sac. Claudin-b morphants exhibited an increase in epithelial permeability to the paracellular marker polyethylene glycol (PEG-4000) and fluorescein isothiocyanate-dextran (FD-4). Accumulation of FD-4 was confined mainly to the yolk sac and pericardial cavity in the claudin-b morphants, suggesting these regions became particularly leaky in the absence of claudin-b expression. Additionally, Na+ efflux was substantially increased in the claudin-b morphants, which contributed to a significant reduction in whole-body Na+ levels. These results indicate that claudin-b normally acts as a paracellular barrier to Na+. Nevertheless, the elevated loss of Na+ in the morphants was compensated by an increase in Na+ uptake. Notably, we observed that the increased Na+ uptake in the morphants was attenuated in the presence of the selective Na+/Cl−-cotransporter (NCC) inhibitor metolazone, or during exposure to Cl−-free water. These results suggested that the increased Na+ uptake in the morphants was, at least in part, mediated by NCC. Furthermore, treatment with an H+-ATPase inhibitor bafilomycin A1 was found to reduce Na+ uptake in the morphants, suggesting that H+-ATPase activity was essential to provide a driving force for Na+ uptake. Overall, the results suggest that claudin-b plays an important role in regulating epithelial permeability and Na+ handling in zebrafish.

scholarly journals Role of internal convection in respiratory gas transfer and aerobic metabolism in larval zebrafish (Danio rerio)

2019 ◽  
Vol 316 (3) ◽  
pp. R255-R264 ◽  
Author(s):  
Malcolm C. Hughes ◽  
Alex M. Zimmer ◽  
Steve F. Perry
Keyword(s):  
Danio Rerio ◽  
Troponin T ◽  
High Surface ◽  
Volume Ratio ◽  
Aerobic Metabolism ◽  
Whole Body ◽  
Gas Transfer ◽  
Larval Zebrafish ◽  
Respiratory Gases ◽  
Internal Convection

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.

Loss of M2 muscarinic receptor function inhibits development of hypoxic bradycardia and alters cardiac β-adrenergic sensitivity in larval zebrafish (Danio rerio)

2009 ◽  
Vol 297 (2) ◽  
pp. R412-R420 ◽  
Author(s):  
Shelby L. Steele ◽  
Kwok Hong Andy Lo ◽  
Vincent Wai Tsun Li ◽  
Shuk Han Cheng ◽  
Marc Ekker ◽  
...  
Keyword(s):  
Heart Rate ◽  
Muscarinic Receptor ◽  
Danio Rerio ◽  
Antisense Oligonucleotides ◽  
Adrenergic Receptor ◽  
Larval Zebrafish ◽  
M2 Muscarinic Receptor ◽  
Β Adrenergic Receptors ◽  
Relative Mrna Expression

Fish exposed to hypoxia develop decreased heart rate, or bradycardia, the physiological significance of which remains unknown. The general muscarinic receptor antagonist atropine abolishes the development of this hypoxic bradycardia, suggesting the involvement of muscarinic receptors. In this study, we tested the hypothesis that the hypoxic bradycardia is mediated specifically by stimulation of the M2 muscarinic receptor, the most abundant subtype in the vertebrate heart. Zebrafish ( Danio rerio) were reared at two levels of hypoxia (30 and 40 Torr Po2) from the point of fertilization. In hypoxic fish, the heart rate was significantly lower than in normoxic controls from 2 to 10 days postfertilization (dpf). At the more severe level of hypoxia (30 Torr Po2), there were significant increases in the relative mRNA expression of M 2 and the cardiac type β-adrenergic receptors ( β1AR, β2aAR, and β2bAR) at 4 dpf. The hypoxic bradycardia was abolished (at 40 Torr Po2) or significantly attenuated (at 30 Torr Po2) in larvae experiencing M2 receptor knockdown (using morpholino antisense oligonucleotides). Sham-injected larvae exhibited typical hypoxic bradycardia in both hypoxic regimens. The expression of β1AR, β2aAR, β2bAR, and M 2 mRNA was altered at various stages between 1 and 4 dpf in larvae experiencing M2 receptor knockdown. Interestingly, M2 receptor knockdown revealed a cardioinhibitory role for the β2-adrenergic receptor. This is the first study to demonstrate a specific role of the M2 muscarinic receptor in the initiation of hypoxic bradycardia in fish.

On the effects of iron ore tailings micro/nanoparticles in embryonic and larval zebrafish (Danio rerio)

2021 ◽  
Vol 759 ◽  
pp. 143456
Author(s):  
Vítor Otacílio de Almeida ◽  
Talita Carneiro Brandão Pereira ◽  
Lilian de Souza Teodoro ◽  
Manuella Escobar ◽  
Carolina Junqueira Ordovás ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Iron Ore ◽  
Larval Zebrafish ◽  
Iron Ore Tailings
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