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

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.

Functional significance and physiological regulation of essential trace metals in fish.

Trace metals such as iron, copper, zinc and manganese play essential roles in various biological processes in fish, including development, energy metabolism and immune response. At embryonic stages, fish obtain essential metals primarily from the yolk, whereas in later life stages (i.e. juvenile and adult), the gastrointestine and the gill are the major sites for the acquisition of trace metals. On a molecular level, the absorption of metals is thought to occur at least in part via specific metal ion transporters, including the divalent metal transporter-1 (DMT1), copper transporter-1 (CTR1), and Zrt- and Irt-like proteins (ZIP). A variety of other proteins are also involved in maintaining cellular and systemic metal homeostasis. Interestingly, the expression and function of these metal transport- and metabolism-related proteins can be influenced by a range of trace metals and major ions. Increasing evidence also demonstrates an interplay between the gastrointestine and the gill for the regulation of trace metal absorption. Therefore, there is a complex network of regulatory and compensatory mechanisms involved in maintaining trace metal balance. Yet, an array of factors is known to influence metal metabolism in fish, such as hormonal status and environmental changes. In this Review, we summarize the physiological significance of iron, copper, zinc and manganese, and discuss the current state of knowledge on the mechanisms underlying transepithelial metal ion transport, metal-metal interactions, and cellular and systemic handling of these metals in fish. Finally, we identify knowledge gaps in the regulation of metal homeostasis and discuss potential future research directions.

Algal Organic Matter Drives Methanogen-Mediated Methylmercury Production in Water from Eutrophic Shallow Lakes.

Algal blooms bring massive amounts of algal organic matter (AOM) into eutrophic lakes, which influences microbial methylmercury (MeHg) production. However, because of the complexity of AOM and its dynamic changes during algal decomposition, the relationship between AOM and microbial Hg methylators remains poorly understood, which hinders predicting MeHg production and its bioaccumulation in eutrophic shallow lakes. To address that, we explored the impacts of AOM on microbial Hg methylators and MeHg production by characterizing dissolved organic matter with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy and quantifying the microbial Hg methylation gene hgcA. We first reveal that the predominance of methanogens, facilitated by eutrophication-induced carbon input, could drive MeHg production in lake water. Specifically, bioavailable components of AOM (i.e., CHONs such as aromatic proteins and soluble microbial byproduct-like materials) increased the abundances (Archaea-hgcA gene: 438-2240% higher) and activities (net CH4 production: 16.0-44.4% higher) of Archaea (e.g., methanogens). These in turn led to enhanced dissolved MeHg levels (24.3-15,918% higher) for three major eutrophic shallow lakes in China. Nevertheless, our model results indicate that AOM-facilitated MeHg production could be offset by AOM-induced MeHg biodilution under eutrophication. Our study would help reduce uncertainties in predicting MeHg production, providing a basis for mitigating the MeHg risk in eutrophic lakes.

Respiratory responses to external ammonia in zebrafish (Danio rerio).

The effects of high external ammonia (HEA) exposure on breathing and the potential involvement of ammonia transporting Rh proteins in ammonia sensing were assessed in larval and adult zebrafish. Acute exposure of adults to either 250 or 500 µM (NH4)2SO4 caused increases in ventilation amplitude (AVENT) without affecting frequency (fVENT), resembling the ventilatory response to hypercapnia rather than hypoxia, during which fVENT was increased exclusively. The hyperventilatory response to HEA was prevented by hyperoxia, indicating that control of breathing through ammonia sensing is likely secondary to O2 chemoreception. Neuroepithelial cells (NECs) isolated from gill filaments exhibited a significant increase of intracellular [Ca2+] in response to 1 mM NH4Cl but this response was small (roughly 30%) compared to the response to hypercapnia (37.5 mmHg; ~800% increase). Immunohistochemistry (IHC) failed to reveal the presence of Rh proteins (Rhcgb, Rhbg or Rhag) in gill filament NECs. Knockout of rhcgb did not affect the ventilatory response of adults to HEA. Larvae at 4 days post fertilization (dpf) responded to HEA with increases in fVENT (AVENT was not measured). The hyperventilatory response of larvae to HEA was attenuated (60% reduction) after treatment from 0 to 4 dpf with the sympathetic neurotoxin 6-hydroxydopamine. In larvae, Rhcgb, Rhbg and Rhag were undetectable by IHC in cutaneous NECs yet the fVENT to HEA following Rhbg knockdown was slightly (22%) attenuated. Thus, the hyperventilatory response to external ammonia in adult zebrafish, while apparently initiated by activation of NECs, does not require Rhcgb, nor is the entry of ammonia into NECs reliant on other Rh proteins. The lack of colocalization of Rh proteins with NECs suggests that the entry of ammonia into NECs in larvae, also is not facilitated by this family of ammonia channels.

Straw return enhances the risks of metals in soil?

Interactions between organic matter (OM) and metals in soils are important natural mechanisms that can mitigate metal bioaccumulation in terrestrial environments. A primary source of OM in soils is straw return, accounting for more than 65% of OM input. Straw-OM has long been believed to reduce metal bioaccumulation, e.g., by immobilizing metals in soils. However, there is growing evidence that straw return could possibly enhance bioavailability and thus risks (i.e., food safety) of some metals in crops, including Cd, Hg, and As. Poor understanding of straw return-induced increases in metal bioavailability would add uncertainty in assessing or mitigating risks of metals in contaminated farming soils. Here, 863 pieces of literature (2000-2019) that reported the effects of straw return on metal bioavailability and bioaccumulation were reviewed. Mechanisms responsible for the increased metal mobility and bioavailability under straw return are summarized, including the effects of dissolution, complexation, and methylation. Effects of straw return on the physiology and the absorption of metals in plants is also discussed (i.e., physiological effect). These mechanisms are then used to explain the observed increases in the mobility, bioavailability, and bioaccumulation of Cd, Hg, and As under straw amendment. Information summarized in this study highlights the importance to re-consider the current straw return policy, particularly in metal-contaminated farmlands.

Influence of Microplastics on the Mobility, Bioavailability, and Toxicity of Heavy Metals: A Review.

Microplastics (MPs) can pose ecological risk to the environment and have the potential to negatively affect human health, raising serious public concerns. It is recognized that MPs could act as a vector for various environmental pollutants including heavy metals and potentially influencing their mobility, fate, and bioavailabilty in the environment. However, knowledge on the mechanisms underpinning the interaction processes between MPs and heavy metals is far from clear. This review discusses the effects of MPs on the adsorption/desorption, speciation and bioavailability, and toxicity of various heavy metals. The present review also systematically identifies the environmental factors (e.g., pH, ionic strength, and organic matters) that could affect their interaction processes. This work aims to establish a meaningful perspective for a comprehensive understanding of the indirect ecological risks of MPs as vectors for contaminants. The work also provides a reference for the development of better regulatory strategies in mitigating the negative effects caused by the co-existence of MPs and heavy metals.

Reassessing the contribution of the Na+/H+ exchanger Nhe3b to Na+ uptake in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing.

Freshwater fishes absorb Na+ from their dilute environment using ion-transporting cells. In larval zebrafish (Danio rerio), Na+ uptake is coordinated by (1) Na+/H+ exchanger 3b (Nhe3b) and (2) H+-ATPase-powered electrogenic uptake in H+-ATPase-rich (HR) cells and by (3) Na+-Cl--cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na+ uptake by larval zebrafish under 'normal' (800 µmol l-1) and 'low' (10 µmol l-1) Na+ conditions. We hypothesized that Na+ uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na+ where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na+ uptake was sustained in nhe3b KO larvae under both Na+ conditions, which led to the exploration of whether compensatory regulation of H+-ATPase or Ncc was responsible for maintaining Na+ uptake in nhe3b KO larvae. mRNA expression of the genes encoding H+-ATPase and Ncc was not altered in nhe3b KO larvae. Moreover, morpholino knockdown of H+-ATPase, which significantly reduced H+ flux by HR cells, did not reduce Na+ uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na+-Cl--cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H+-ATPase morpholino and chloride-free conditions did not reduce Na+ uptake under normal or low Na+ These findings highlight the flexibility of the Na+ uptake system and demonstrate that Nhe3b is expendable to Na+ uptake in zebrafish and that our understanding of Na+ uptake mechanisms in this species is incomplete.

Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing?

Loss-of-function technologies, such as morpholino- and RNAi-mediated gene knockdown, and TALEN- and CRISPR/Cas9-mediated 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).

Assessing the role of the acid-sensing ion channel ASIC4b in sodium uptake by larval zebrafish.

Na+ uptake in larval zebrafish (Danio rerio) is coordinated by three mechanisms: Na+/H+-exchanger 3b (NHE3b) expressed in H+-ATPase-rich (HR) cells, an unidentified Na+ channel coupled to electrogenic H+-ATPase expressed in HR cells, and Na+-Cl--cotransporter (NCC) expressed in NCC cells. Recently, acid-sensing ion channels (ASICs) were proposed to be the putative Na+ channel involved in H+-ATPase-mediated Na+ uptake in adult zebrafish and rainbow trout. In the present study, we hypothesized that ASICs also play this role in Na+ uptake in larval zebrafish. In support of this hypothesis, immunohistochemical analyses revealed that ASIC4b was expressed in HR cells on the yolk sac skin at 4 days post-fertilization (dpf). However, neither treatment with the ASIC-specific blocker 4,6-diamidino-2-phenylindole (DAPI) nor morpholino knockdown of ASIC4b reduced Na+ uptake in circumneutral conditions at 4 dpf. However, because ASIC4b knockdown led to significant increases in the mRNA expression of nhe3b and ncc and a significant increase in HR cell density, it is possible that Na+ influx was sustained by increased participation of non-ASIC4b pathways. Moreover, when fish were reared in acidic water (pH = 4), ASIC4b knockdown led to a stimulation of Na+ uptake at 3 and 4 dpf, results which also were inconsistent with an essential role for ASIC-mediated Na+ uptake, even under conditions known to constrain Na+ uptake via NHE3b. Thus, while ASIC4b clearly is expressed in HR cells, the current functional experiments cannot confirm its involvement in Na+ uptake in larval zebrafish.

Zebrafish as a Model System for Investigating the Compensatory Regulation of Ionic Balance during Metabolic Acidosis.

Zebrafish (Danio rerio) have become an important model for integrative physiological research. Zebrafish inhabit a hypo-osmotic environment; to maintain ionic and acid-base homeostasis, they must actively take up ions and secrete acid to the water. The gills in the adult and the skin at larval stage are the primary sites of ionic regulation in zebrafish. The uptake of ions in zebrafish is mediated by specific ion transporting cells termed ionocytes. Similarly, in mammals, ion reabsorption and acid excretion occur in specific cell types in the terminal region of the renal tubules (distal convoluted tubule and collecting duct). Previous studies have suggested that functional regulation of several ion transporters/channels in the zebrafish ionocytes resembles that in the mammalian renal cells. Additionally, several mechanisms involved in regulating the epithelial ion transport during metabolic acidosis are found to be similar between zebrafish and mammals. In this article, we systemically review the similarities and differences in ionic regulation between zebrafish and mammals during metabolic acidosis. We summarize the available information on the regulation of epithelial ion transporters during acidosis, with a focus on epithelial Na⁺, Cl- and Ca2+ transporters in zebrafish ionocytes and mammalian renal cells. We also discuss the neuroendocrine responses to acid exposure, and their potential role in ionic compensation. Finally, we identify several knowledge gaps that would benefit from further study.

A role for sodium-chloride cotransporters in the rapid regulation of ion uptake following acute environmental acidosis: new insights from the zebrafish model.

The effects of acute exposure to acidic water on Na+ and Cl- homeostasis, and the mechanisms underlying their compensatory regulation, were investigated in the larval zebrafish Danio rerio Exposure to acidic water (pH 4.0; control pH 7.6) for 2 h significantly reduced Na+ uptake and whole body Na+ content. Nevertheless, the capacity for Na+ uptake was substantially increased in fish preexposed to acidic water but measured in control water. Based on the accumulation of the Na+-selective dye, Sodium Green, two ionocyte subtypes exhibited intracellular Na+ enrichment after preexposure to acidic water: H+-ATPase rich (HR) cells, which coexpress the Na+/H+ exchanger isoform 3b (NHE3b), and a non-HR cell population. In fish experiencing Na+-Cl- cotransporter (NCC) knockdown, we observed no Sodium Green accumulation in the latter cell type, suggesting the non-HR cells were NCC cells. Elimination of NHE3b-expressing HR cells did not prevent the increased Na+ uptake following acid exposure. On the other hand, the increased Na+ uptake was abolished when the acidic water was enriched with Na+ and Cl-, but not with Na+ only, indicating that the elevated Na+ uptake after acid exposure was associated with the compensatory regulation of Cl- Further examinations demonstrated that acute acid exposure also reduced whole body Cl- levels and increased the capacity for Cl- uptake. Moreover, knockdown of NCC prevented the increased uptake of both Na+ and Cl- after exposure to acidic water. Together, the results of the present study revealed a novel role of NCC in the compensatory regulation of Na+ and Cl- uptake following acute acidosis.

Inhibition of calcium uptake during hypoxia in developing zebrafish is mediated by hypoxia-inducible factor.

The present study investigated the potential role of hypoxia-inducible factor (HIF) in calcium homeostasis in developing zebrafish (Danio rerio). It was demonstrated that zebrafish raised in hypoxic water (30 mmHg; control, 155 mmHg PO2 ) until 4 days post-fertilization exhibited a substantial reduction in whole-body Ca2+ levels and Ca2+ uptake. Ca2+ uptake in hypoxia-treated fish did not return to pre-hypoxia (control) levels within 2 h of transfer back to normoxic water. Results from real-time PCR showed that hypoxia decreased the whole-body mRNA expression levels of the epithelial Ca2+ channel (ecac), but not plasma membrane Ca2+-ATPase (pmca2) or Na+/Ca2+-exchanger (ncx1b). Whole-mount in situ hybridization revealed that the number of ecac-expressing ionocytes was reduced in fish raised in hypoxic water. These findings suggested that hypoxic treatment suppressed the expression of ecac, thereby reducing Ca2+ influx. To further evaluate the potential mechanisms for the effects of hypoxia on Ca2+ regulation, a functional gene knockdown approach was employed to prevent the expression of HIF-1αb during hypoxic treatment. Consistent with a role for HIF-1αb in regulating Ca2+ balance during hypoxia, the results demonstrated that the reduction of Ca2+ uptake associated with hypoxic exposure was not observed in fish experiencing HIF-1αb knockdown. Additionally, the effects of hypoxia on reducing the number of ecac-expressing ionocytes was less pronounced in HIF-1αb-deficient fish. Overall, the current study revealed that hypoxic exposure inhibited Ca2+ uptake in developing zebrafish, probably owing to HIF-1αb-mediated suppression of ecac expression.

Neuroendocrine control of ionic balance in zebrafish.

Zebrafish (Danio rerio) is an emerging model for integrative physiological research. In this mini-review, we discuss recent advances in the neuroendocrine control of ionic balance in this species, and identify current knowledge gaps and issues that would benefit from further investigation. Zebrafish inhabit a hypo-ionic environment and therefore are challenged by a continual loss of ions to the water. To maintain ionic homeostasis, they must actively take up ions from the water and reduce passive ion loss. The adult gill or the skin of larvae are the primary sites of ionic regulation. Current models for the uptake of major ions in zebrafish incorporate at least three types of ion transporting cells (also called ionocytes); H(+)-ATPase-rich cells for Na(+) uptake, Na(+)/K(+)-ATPase-rich cells for Ca(2+) uptake, and Na(+)/Cl(-)-cotransporter expressing cells for both Na(+) and Cl(-) uptake. The precise molecular mechanisms regulating the paracellular loss of ions remain largely unknown. However, epithelial tight junction proteins, including claudins, are thought to play a critical role in reducing ion losses to the surrounding water. Using the zebrafish model, several key neuroendocrine factors were identified as regulators of epithelial ion movement, including the catecholamines (adrenaline and noradrenaline), cortisol, the renin-angiotensin system, parathyroid hormone and prolactin. Increasing evidence also suggests that gasotransmitters, such as H2S, are involved in regulating ion uptake.

Hydrogen sulfide promotes calcium uptake in larval zebrafish.

Hydrogen sulfide (H2S) can act as a signaling molecule for various ion channels and/or transporters; however, little is known about its potential involvement in Ca(2+) balance. Using developing zebrafish (Danio rerio) as an in vivo model system, the present study demonstrated that acute exposure to H2S donors increased Ca(2+) influx at 4 days postfertilization, while chronic (3-day) exposure caused a rise in whole body Ca(2+) levels. The mRNA expression of Ca(2+)-transport-related genes was unaffected by H2S exposure, suggesting that posttranscriptional modifications were responsible for the altered rates of Ca(2+) uptake. Indeed, treatment of fish with the protein kinase A inhibitor H-89 abolished the H2S-mediated stimulation of Ca(2+) influx, suggesting that H2S increased Ca(2+) influx by activating cAMP-protein kinase A pathways. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are two key enzymes in the endogenous synthesis of H2S. Using an antisense morpholino knockdown approach, we demonstrated that Ca(2+) influx was reduced in CBS isoform b (CBSb)- but not in CSE-deficient fish. Interestingly, the reduction in Ca(2+) influx in CBSb-deficient fish was observed only in fish that were acclimated to low-Ca(2+) water (i.e., 25 µM Ca(2+); control: 250 µM Ca(2+)). Similarly, mRNA expression of cbsb but not cse was increased in fish acclimated to low-Ca(2+) water. Results from whole-mount immunohistochemistry further revealed that CBSb was expressed in Na(+)-K(+)-ATPase-rich cells, which are implicated in Ca(2+) uptake in zebrafish larvae. Collectively, the present study suggests a novel role for H2S in promoting Ca(2+) influx, particularly in a low-Ca(2+) environment.

A role for transcription factor glial cell missing 2 in Ca2+ homeostasis in zebrafish, Danio rerio.

The present study investigated the role of the transcription factor, glial cell missing 2 (gcm2), in Ca(2+) regulation in zebrafish larvae. Translational gene knockdown of gcm2 decreased Ca(2+) uptake and the density of ionocytes expressing the epithelial Ca(2+) channel (ecac), and disrupted the overall Ca(2+) balance. Ca(2+) uptake and the expression of gcm2 messenger RNA (mRNA) were significantly elevated in larvae acclimated to low Ca(2+) water (25 µM); the stimulation of Ca(2+) uptake was not observed in fish experiencing gcm2 knockdown. Acclimation to acidic water (pH 4) significantly reduced whole-body Ca(2+) content owing to reduced Ca(2+) uptake and increased Ca(2+) efflux. However, ecac mRNA levels and the density of ecac-expressing ionocytes were increased in fish acclimated to acidic water, and maximal Ca(2+) uptake capacity (J MAX) was significantly increased when measured in control water (pH ~7.4). Acclimation of larvae to acidic water significantly increased gcm2 mRNA expression, and in gcm2 morphants, no such stimulation in Ca(2+) uptake was observed after their return to control water. Overexpression of gcm2 mRNA resulted in a significant increase in the numbers of ecac-expressing ionocytes and Ca(2+) uptake. These observations reveal a critical role for gcm2 in Ca(2+) homeostasis in zebrafish larvae.

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

The present study investigated the role of hydrogen sulfide (H2S) in regulating Na(+) uptake in larval zebrafish, Danio rerio. Waterborne treatment of larvae at 4 days post-fertilization (dpf) with Na2S or GYY-4137 (chemicals known to generate H2S) significantly reduced Na(+) uptake. Exposure of larvae to water enriched with NaCl (1 mM NaCl) caused a pronounced reduction in Na(+) uptake which was prevented by pharmacological inhibition of cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE), two key enzymes involved in the endogenous synthesis of H2S. Furthermore, translational gene knockdown of CSE and CBSb significantly increased the basal rate of Na(+) uptake. Waterborne treatment with Na2S significantly decreased whole-body acid excretion and reduced Na(+) uptake in larval zebrafish preexposed to acidic (pH 4.0) water (a condition shown to promote Na(+) uptake via Na(+)-H(+)-exchanger 3b, NHE3b). However, Na2S did not affect Na(+) uptake in larvae depleted of NHE3b-containing ionocytes (HR cells) after knockdown of transcription factor glial cell missing 2 (gcm2) in which Na(+) uptake occurs predominantly via Na(+)-Cl(-) co-transporter (NCC)-containing cells. These observations suggest that Na(+) uptake via NHE3b, but not NCC, is regulated by H2S. Whole-mount immunohistochemistry demonstrated that ionocytes expressing NHE3b also express CSE. These data suggests a physiologically relevant role of H2S as a mechanism to lower Na(+) uptake in zebrafish larvae, probably through its inhibitory action on NHE3b.

The water channel aquaporin-1a1 facilitates movement of CO2 and ammonia in zebrafish (Danio rerio) larvae.

The present study tested the hypothesis that zebrafish (Danio rerio) aquaporin-1a1 (AQP1a1) serves as a multi-functional channel for the transfer of the small gaseous molecules, CO2 and ammonia, as well as water, across biological membranes. Zebrafish embryos were microinjected with a translation-blocking morpholino oligonucleotide targeted to AQP1a1. Knockdown of AQP1a1 significantly reduced rates of CO2 and ammonia excretion, as well as water fluxes, in larvae at 4 days post fertilization (dpf). Because AQP1a1 is expressed both in ionocytes present on the body surface and in red blood cells, the haemolytic agent phenylhydrazine was used to distinguish between the contributions of AQP1a1 to gas transfer in these two locations. Phenylhydrazine treatment had no effect on AQP1a1-linked excretion of CO2 or ammonia, providing evidence that AQP1a1 localized to the yolk sac epithelium, rather than red blood cell AQP1a1, is the major site of CO2 and ammonia movements. The possibility that AQP1a1 and the rhesus glycoprotein Rhcg1, which also serves as a dual CO2 and ammonia channel, act in concert to facilitate CO2 and ammonia excretion was explored. Although knockdown of each protein did not affect the abundance of mRNA and protein of the other protein under control conditions, impairment of ammonia excretion by chronic exposure to high external ammonia triggered a significant increase in the abundance of AQP1a1 mRNA and protein in 4 dpf larvae experiencing Rhcg1 knockdown. Collectively, these results suggest that AQP1a1 in zebrafish larvae facilitates the movement of CO2 and ammonia, as well as water, in a physiologically relevant fashion.

A role for nitric oxide in the control of breathing in zebrafish (Danio rerio).

Nitric oxide (NO) is a gaseous neurotransmitter, which, in adult mammals, modulates the acute hypoxic ventilatory response; its role in the control of breathing in fish during development is unknown. We addressed the interactive effects of developmental age and NO in the control of piscine breathing by measuring the ventilatory response of zebrafish (Danio rerio) adults and larvae to NO donors and by inhibiting endogenous production of NO. In adults, sodium nitroprusside (SNP), a NO donor, inhibited ventilation; the extent of the ventilatory inhibition was related to the pre-existing ventilatory drive, with the greatest inhibition exhibited during exposure to hypoxia (PO2=5.6 kPa). Inhibition of endogenous NO production using L-NAME suppressed the hypoventilatory response to hyperoxia, supporting an inhibitory role of NO in adult zebrafish. Neuroepithelial cells (NECs), the putative oxygen chemoreceptors of fish, contain neuronal nitric oxide synthase (nNOS). In zebrafish larvae at 4 days post-fertilization, SNP increased ventilation in a concentration-dependent manner. Inhibition of NOS activity with L-NAME or knockdown of nNOS inhibited the hypoxic (PO2=3.5 kPa) ventilatory response. Immunohistochemistry revealed the presence of nNOS in the NECs of larvae. Taken together, these data suggest that NO plays an inhibitory role in the control of ventilation in adult zebrafish, but an excitatory role in larvae.

The role of hydrogen sulphide in the control of breathing in hypoxic zebrafish (Danio rerio).

The current study investigated the role of hydrogen sulphide (H2S) in oxygen sensing, intracellular signalling and promotion of ventilatory responses to hypoxia in adult and larval zebrafish (Danio rerio). Both larval and adult zebrafish exhibited a dose-dependent increase in ventilation to sodium sulphide (Na2S), an H2S donor. In vertebrates, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are enzymes that catalyse the endogenous production of H2S. In adult zebrafish, inhibition of both CBS and CSE with aminooxyacetate (AOA) and propargyl glycine (PPG) blunted or abolished the hypoxic hyperventilation, and the addition of Na2S to the water partially rescued the effects of inhibiting endogenous H2S production. In zebrafish larvae (4 days post-fertilization), gene knockdown of either CBS or CSE using morpholinos attenuated the hypoxic ventilatory response. Furthermore, the intracellular calcium concentration of isolated neuroepithelial cells (NECs), which are putative oxygen chemoreceptors, increased significantly when these cells were exposed to 50 µm Na2S, supporting a role for H2S in Ca(2+)-evoked neurotransmitter release in these cells. Finally, immunohistochemical labelling showed that NECs dissociated from adult gill contained CBS and CSE, whereas cutaneous NECs in larval zebrafish expressed only CSE. Taken together, these data show that H2S can be produced in the putative oxygen-sensing cells of zebrafish, the NECs, in which it appears to play a pivotal role in promoting the hypoxic ventilatory response.