Alternation of social behaviors for zebrafish (Danio rerio) in response to acute cold stress.

Low temperature is one of the most common abiotic stresses for aquatic ectotherms. Ambient low temperatures reduce the metabolic rate of teleosts, therefore, teleosts have developed strategies to modulate their physiological status for energy saving in response to cold stress, including behaviors, circulatory system, respiratory function, and metabolic adjustments. Many teleosts are social animals and they can live in large schools to serve a variety of functions, including predator avoidance, foraging efficiency, and reproduction. However, the impacts of acute cold stress on social behaviors of fish remain unclear. In the present study, we test the hypothesis that zebrafish alter their social behaviors for energy saving as a strategy in response to acute cold stress. We found that acute cold stress increased shoaling behavior that reflected a save-energy strategy for fish to forage and escape from the predators under cold stress. The aggressive levels measured by fighting behavior tests and mirror fighting tests were reduced by cold treatment. In addition, we also found that acute cold stress impaired the learning ability but did not affect memory. Our findings provided evidence that acute cold stress alters the social behaviors of aquatic ectotherms for energy saving; knowledge of their responses to cold is essential for their conservation and management.

Sublethal ammonia induces alterations of emotions, cognition, and social behaviors in zebrafish (Danio rerio).

Ammonia pollutants were usually found in aquatic environments is due to urban sewage, industrial wastewater discharge, and agricultural runoff and concentrations as high as 180 mg/L (NH4+) have been reported in rivers. High ammonia levels are known to impair multiple tissue and cell functions and cause fish death. Although ammonia is a potent neurotoxin, how sublethal concentrations of ammonia influence the central nervous system (CNS) and the complex behaviors of fish is still unclear. In the present study, we demonstrated that acute sublethal ammonia exposure can change social behavior of adult zebrafish. The exposure to 90 mg /L of (NH4+) for 4 h induced a strong fear response and lower shoaling cohesion; exposure to 180 mg /L of (NH4+) for 4 h reduced the aggressiveness, and social recognition, while the anxiety, social preference, learning, and short-term memory were not affected. Messenger RNA expressions of glutaminase and glutamate dehydrogenase in the brain were induced, suggesting that ammonia exposure altered glutamate neurotransmitters in the CNS. Our findings in zebrafish provided delicate information of ammonia neurotoxicity in complex higher-order social behaviors, which has not been revealed previously. In conclusion, sublethal and acute ammonia exposure can change specific behaviors of fish, which might lead to reductions in individual and population fitness levels.

Transient receptor potential vanilloid 4 modulates ion balance through the isotocin pathway in zebrafish (Danio rerio).

Isotocin controls ion regulation through modulating the functions of ionocytes (also called mitochondria-rich cells or chloride cells). However, little is known about the upstream molecule of the isotocin system. Herein, we identify transient receptor potential vanilloid 4 (TRPV4), which regulates the mRNA and protein expressions of isotocin and affects ion regulation through the isotocin pathway. Double immunohistochemical results showed that TRPV4 is expressed in isotocinergic neurons in the hypothalamus of the adult zebrafish brain. To further elucidate the roles of TRPV4, we manipulated TRPV4 protein expression and evaluated its ionoregulatory functions in zebrafish embryos. TRPV4 gene knockdown with morpholino oligonucleotides decreased ionic contents (Na+, Cl-, and Ca2+) of whole larvae and the H+-secreting function of larval skin of zebrafish. mRNA expressions of ionocyte-related transporters, including H+-ATPase, the epithelial Ca2+ channel, and the Na+-Cl- cotransporter, were also suppressed in trpv4 morphants. Numbers of ionocytes (H+-ATPase-rich cells and Na+-K+-ATPase-rich cells) and epidermal stem cells in zebrafish larval skin also decreased after trpv4 knockdown. Our results showed that TRPV4 modulates ion balance through the isotocin pathway.

Oestrogen-related receptor α is required for transepithelial H+ secretion in zebrafish.

Oestrogen-related receptor α (ERRα) is an orphan nuclear receptor which is important for adaptive metabolic responses under conditions of increased energy demand, such as cold, exercise and fasting. Importantly, metabolism under these conditions is usually accompanied by elevated production of organic acids, which may threaten the body acid-base status. Although ERRα is known to help regulate ion transport by the renal epithelia, its role in the transport of acid-base equivalents remains unknown. Here, we tested the hypothesis that ERRα is involved in acid-base regulation mechanisms by using zebrafish as the model to examine the effects of ERRα on transepithelial H(+) secretion. ERRα is abundantly expressed in H(+)-pump-rich cells (HR cells), a group of ionocytes responsible for H(+) secretion in the skin of developing embryos, and its expression is stimulated by acidic (pH 4) environments. Knockdown of ERRα impairs both basal and low pH-induced H(+) secretion in the yolk-sac skin, which is accompanied by decreased expression of H(+)-secreting-related transporters. The effect of ERRα on H(+) secretion is achieved through regulating both the total number of HR cells and the function of individual HR cells. These results demonstrate, for the first time, that ERRα is required for transepithelial H(+) secretion for systemic acid-base homeostasis.

Investigation of ammonia-induced lethal toxicity toward ion regulation in zebrafish embryos.

Ammonia is an environmental pollutant that is toxic to all aquatic animals. However, the mechanism of ammonia toxicity toward the ion regulatory function of early-stage fish has not been fully documented. We addressed this issue using zebrafish embryos as a model. We hypothesized that ammonia might impair ion regulation by inducing oxidative stress, mitochondrial dysfunction, and cell death of epidermal ionocytes and keratinocytes in zebrafish embryos. After exposure to various concentrations (10- 30 mM) of NH4Cl for 96 h, mortality increased up to 50 % and 100 % at 25 and 30 mM, respectively. Whole-embryo sodium, potassium, and calcium contents decreased at ≥10 mM, suggesting dysfunction of ion regulation. Numbers of H+-ATPase-rich (HR) cells and Na+/K+-ATPase-rich (NaR) cells (two ionocyte subtypes) were not significantly altered at 15 or 20 mM, while the mitochondrial abundance significantly decreased and reactive oxygen species (ROS) levels significantly increased in ionocytes. Moreover, caspase-3-dependent apoptosis was found in epidermal keratinocytes. Whole-embryo transcript levels of several genes involved in ion regulation, antioxidation, and apoptosis were upregulated after ammonia exposure. In conclusion, ammonia exposure was shown to induce oxidative stress and mitochondrial dysfunction in ionocytes and apoptosis in keratinocytes, thereby impairing ion regulation and ultimately leading to the death of zebrafish embryos.

Ammonia exposure impairs bone mineralization in zebrafish (Danio rerio) larvae.

Ammonia is a major pollutant of freshwater environments. Previous studies have indicated that ammonia exposure adversely affects the physiology of freshwater fish. However, its effect on bone mineralization in freshwater fish larvae remains unclear. In this study, zebrafish larvae were used as a model to investigate the effects of different ammonia levels (0, 2.5, 5, and 10 mM NH4Cl) on the survival rate, body length, and bone mineralization of fish. The survival rate of zebrafish embryos exposed to different NH4Cl concentrations for 8 days was not affected. In contrast, the body length and bone mineralization of zebrafish larvae at 8 days post fertilization (dpf) were significantly reduced at 5 and 10 mM NH4Cl exposure. Further investigations revealed that ammonia exposure decreased the mRNA expression of osteoblast-related genes and increased that of osteoclast-related genes. Additionally, exposure to 5 mM and 10 mM NH4Cl induced the production of reactive oxygen species (ROS). 10 mM-but not 5 mM-NH4Cl exposure reduced the calcium and phosphorus content in 8 dpf zebrafish larvae. In conclusion, ammonia exposure induces bone resorption, while decreasing the calcium and phosphorus content of the whole body and bone formation, resulting in impaired bone mineralization in fish larvae.

Conserved expression of the zebrafish syt4 gene in GABAergic neurons in the cerebellum of adult fishes revealed by mammalian SYT4 immunoreactive-like signals.

Synaptotagmin 4 (syt4) belongs to the synaptotagmin protein family, which has 17 and 28 family members in human and zebrafish, respectively. In zebrafish and rodents, syt4 is known to express abundantly in the entire central nervous system in the early developmental stages. In adult rodents, the gene expression shifts to be predominant in the cerebellum, mostly in Purkinje cells, a type of GABAergic neurons. However, there is no report of the expression pattern of syt4 in the adult zebrafish brain. Therefore, we hypothesize that the expression of syt4 is conserved in adult zebrafish and is specific to the GABAergic neurons, likely Purkinje cells, in the cerebellum. To examine the hypothesis, we first show that only one copy of syt4 gene remains in the zebrafish genome, and it is orthologous to the gene in other vertebrates. We further observe mammalian SYT4 antibody immunoreactive-like (mSYT4-ir) signals in several structures in the hindbrain including the medial divisions of the valvula cerebelli and the corpus cerebelli. In addition, our observations indicate the presence of mSYT4-ir signals in GABAergic neurons, most notably in the Purkinje cell layer of the molecular layer in the aforementioned structures. Conversely, mSYT4-ir signals are not observed in glutamatergic or cholinergic neurons. Therefore, we deduce that the syt4 gene in zebrafish exhibits a homologous expression pattern to those of previously studied vertebrate species, which is revealed by the positive immunoreactive-like signals of mammalian SYT4 antibodies.

Proton-facilitated ammonia excretion by ionocytes of medaka (Oryzias latipes) acclimated to seawater.

The proton-facilitated ammonia excretion is critical for a fish's ability to excrete ammonia in freshwater. However, it remains unclear whether that mechanism is also critical for ammonia excretion in seawater (SW). Using a scanning ion-selective electrode technique (SIET) to measure H(+) gradients, an acidic boundary layer was detected at the yolk-sac surface of SW-acclimated medaka (Oryzias latipes) larvae. The H(+) gradient detected at the surface of ionocytes was higher than that of keratinocytes in the yolk sac. Treatment with Tricine buffer or EIPA (a NHE inhibitor) reduced the H(+) gradient and ammonia excretion of larvae. In situ hybridization and immunochemistry showed that slc9a2 (NHE2) and slc9a3 (NHE3) were expressed in the same SW-type ionocytes. A real-time PCR analysis showed that transfer to SW downregulated branchial mRNA expressions of slc9a3 and Rhesus glycoproteins (rhcg1, rhcg2, and rhbg) but upregulated that of slc9a2. However, slc9a3, rhcg1, rhcg2, and rhbg expressions were induced by high ammonia in SW. This study suggests that SW-type ionocytes play a role in acid and ammonia excretion and that the Na(+)/H(+) exchanger and Rh glycoproteins are involved in the proton-facilitated ammonia excretion mechanism.

Spatial expression and functional flexibility of monocarboxylate transporter isoforms in the zebrafish brain.

The present study provides in vivo evidence to prove the functional plasticity of monocarboxylate transporters (MCTs) in brains of vertebrates using zebrafish (Danio rerio) as a model. In the mammalian central nervous system (CNS), energy demands are largely met by oxidation of glucose. In recent studies, in addition to glucose, lactate is also considered an energy substrate for the CNS. Astrocytes were demonstrated to play an important role in transporting lactate as metabolic substrate from capillaries to neurons through monocarboxylate transporters (MCTs). The present study was to use zebrafish as an in vivo model to test the hypothesis of whether the various MCT homologs play differential roles in the development and functioning of the CNS. Using RT-PCR and double in situ hybridization coupling with immunocytochemical staining experiments, zebrafish MCTs1-4 were all found to be expressed in brains of embryos, and were further elucidated to be localized in both neurons and astrocytes. Loss-of-functions by morpholino knockdown further provided in vivo evidences to infer that zMCTs1, -2, and -4 may be involved in metabolite transport and functioning in the developing brain. Subsequent rescue experiments with capped mRNAs of specific isoforms further indicated that zMCT2 is an indispensable monocarboxylate-transporting route for CNS development and function in zebrafish. This information is essential for identifying proper candidates of MCT isoforms that are involved in the development and functioning of the CNS.

Fenpropathrin causes alterations in locomotion and social behaviors in zebrafish (Danio rerio).

Fenpropathrin is one of the widely used pyrethroid pesticides in agriculture and is frequently detected in the environment, groundwater, and food. While fenpropathrin was found to have neurotoxic effects in mammals, it remains unclear whether it has similar effects on fish. Here, we used adult zebrafish to investigate the impacts of fenpropathrin on fish social behaviors and neural activity. Exposure of adult zebrafish to 500 ppb of fenpropathrin for 72 h increased anxiety levels but decreased physical fitness, as measured by a novel tank diving test and swimming tunnel test. Fish exposed to fenpropathrin appeared to spend more time in the conspecific zone of the tank, possibly seeking greater comfort from their companions. Although learning, memory, and aggressive behavior did not change, fish exposed to fenpropathrin appeared to have shorter fighting durations. The immunocytochemical results showed the tyrosine hydroxylase antibody-labeled dopaminergic neurons in the teleost posterior tuberculum decreased in the zebrafish brain. According to a quantitative polymerase chain reaction (qPCR) analysis of the brain, exposure to fenpropathrin resulted in a decrease in the messenger (m)RNA expression of monoamine oxidase (mao), an enzyme that facilitates the deamination of dopamine. In contrast, the mRNA expression of the sncga gene, which may trigger Parkinson's disease, was found to have increased. There were no changes observed in expressions of genes related to antioxidants and apoptosis between the control and fenpropathrin-exposed groups. We provide evidence to demonstrate the defect of the neurotoxicity of fenpropathrin toward dopaminergic neurons in adult zebrafish.

Retention of ion channel genes expression increases Japanese medaka survival during seawater reacclimation.

Euryhaline teleosts exhibit varying acclimability to survive in environments that alternate between being hypotonic and hypertonic. Such ability is conferred by ion channels expressed by ionocytes, the ion-regulating cells in the gills or skin. However, switching between environments is physiologically challenging, because most channels can only perform unidirectional ion transportation. Coordination between acute responses, such as gene expression, and long-term responses, such as cell differentiation, is believed to strongly facilitate adaptability. Moreover, the pre-acclimation to half seawater salinity can improve the survivability of Japanese medaka (Oryzias latipes) during direct transfer to seawater; here, the ionocytes preserve hypertonic acclimability while performing hypotonic functions. Whether acclimability can be similarly induced in a closed species and their corresponding responses in terms of ion channel expression remain unclear. In the present study, Japanese medaka pre-acclimated in brackish water were noted to have higher survival rates while retaining higher expression of the three ion channel genes ATP1a1a.1, ATP1b1b, and SLC12a2a. This retention was maintained up to 2 weeks after the fish were transferred back into freshwater. Notably, this induced acclimability was not found in its close kin, Indian medaka (Oryzias dancena), the natural habitat of which is brackish water. In conclusion, Japanese medaka surpassed Indian medaka in seawater acclimability after experiencing exposure to brackish water, and this ability coincided with seawater-retention gene expression.

Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water.

To investigate whether Na(+) uptake by zebrafish is dependent on NH4(+) excretion, a scanning ion-selective electrode technique was applied to measure Na(+) and NH4(+) gradients at the yolk-sac surface of zebrafish larvae. Low-Na(+) acclimation induced an inward Na(+) gradient (uptake), and a combination of low Na(+) and high NH4(+) induced a larger inward Na(+) gradient. When measuring the ionic gradients, raising the external NH4(+) level (5 mM) blocked NH4(+) excretion and Na(+) uptake; in contrast, raising the external Na(+) level (10 mM) simultaneously enhanced Na(+) uptake and NH4(+) excretion. The addition of MOPS buffer (5 mM), which is known to block NH4(+) excretion, also suppressed Na(+) uptake. These results showed that Na(+) uptake and NH4(+) excretion by larval skin are associated when ambient Na(+) level is low. Knockdown of Rhcg1 translation with morpholino-oligonucleotides decreased both NH4(+) excretion and Na(+) uptake by the skin and Na(+) content of whole larvae. Knockdown of nhe3b translation or inhibitor (5-ethylisopropyl amiloride) treatment also decreased both the NH4(+) excretion and Na(+) uptake. This study provides loss-of-function evidence for the involvement of Rhcg1 and NHE3b in the ammonium-dependent Na(+) uptake mechanism in zebrafish larvae subjected to low-Na(+) water.

WITHDRAWN: Spatial expression and functional flexibility of monocarboxylate transporter isoforms in the zebrafish brain.

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Role of the Basolateral Na+/H+ Exchanger-2 (NHE2) in Ionocytes of Seawater- Acclimated Medaka (Oryzias latipes).

Ionocytes in the skin and gills of seawater (SW) fishes are responsible for acid-base regulation and salt secretion. Na+/H+ exchangers (NHEs) are considered the major acid (H+)-secreting transporters in ionocytes of SW fishes. However, the subcellular localization and function of a specific NHE isoform (NHE2) have never clearly been revealed. In this study, we cloned and sequenced NHE2 from an SW-acclimated medaka (Oryzias latipes) and examined its functions in medaka embryos. A phylogenetic analysis showed that the evolutionary relationships of mammalian NHE2 and NHE4 are close to those of fish NHE2. A gene structure analysis showed that tetrapod NHE4 might be a tandem duplication of fish NHE2. Immunohistochemistry with a medaka-specific antibody localized NHE2 to the basolateral membrane of ionocytes. Lost-of-function experiments with photo-activated morpholino oligonucleotides showed that both H+ and Cl- secretion by ionocytes were suppressed in NHE2-knockdown embryos, suggesting that the basolateral NHE2 facilitates acid and salt secretion by ionocytes of medaka in seawater.

Acute exposure to polystyrene nanoplastics impairs skin cells and ion regulation in zebrafish embryos.

The presence of nanoplastics in aquatic environments is a global problem. Accumulating evidence shows that nanoplastics can accumulate in fish and influence internal organs. However, it is still unknown if nanoplastics can impair skin cells (keratinocytes and ionocytes), which play critical roles in maintaining body fluid homeostasis. In the present study, zebrafish embryos were exposed to polystyrene nanoplastics (PS-NPs; 25 nm in size, at 0, 10, 25, and 50 mg/L) for 96 h to test the effects of PS-NPs on skin functions. After exposure to 50 mg/L, the survival rate, ion (Na+, K+, and Ca2+) contents, and acid/ammonia excretion by skin cells of embryos significantly declined. The apical structure of skin keratinocytes was damaged at 10, 25, and 50 mg/L. The number and mitochondrial activity of ionocytes were reduced at 25 and 50 mg/L. Reactive oxygen species (ROS) levels indicated by CellROX staining showed that both ionocytes and keratinocytes were under oxidative stress. PS-NPs reduced the mRNA expression of antioxidant genes (sod1, sod2, cat, and gpx1a), and promoted apoptosis-related genes (casp3a). Taken together, this study suggests that PS-NPs might suppress antioxidative reactions and induce oxidative stress, leading to mitochondrial damage and cell death of ionocytes, eventually impairing skin functions including ion uptake, pH regulation, and ammonia excretion.

Differential effects of silver nanoparticles on two types of mitochondrion-rich ionocytes in zebrafish embryos.

Silver nanoparticles (AgNPs) are increasingly used in our daily life and have become a potential environmental hazard. However, the toxic effects of AgNPs on the early stages of fish are not fully understood, and little is known about their effects on specific types of ionocytes. Using zebrafish embryos as a model, this study examined the effects (changes in cell number, morphology, NH4+ secretion and gene expression) of sublethal concentrations of AgNPs (0.1, 1, and 3 mg/L) on two major types of ionocytes: H+ pump-rich (HR) ionocytes, and Na+ pump-rich (NaR) ionocytes in the skin of embryos. After exposure to AgNPs for 96 h, the number of HR ionocytes significantly declined by 30% and 41% in the 1 and 3 mg/L AgNP groups, respectively. In addition, the apical opening of HR ionocytes became smaller, suggesting that AgNPs impaired the critical structure for ion transport. NH4+ secretion by HR ionocytes of embryos also declined significantly after AgNP exposure. In contrast, the number of NaR ionocytes increased by 29% and 43% in the 1 and 3 mg/L AgNP groups, respectively, while these cells deformed their shape. AgNPs altered mRNA levels of several ion channel and transporter genes involved in the functions of HR ionocytes and NaR ionocytes, and influenced hormone genes involved in regulating calcium homeostasis. This study shows that AgNPs can cause differential adverse effects on two types of ionocytes and the effects can threaten fish survival.

Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae.

In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H(+), Na(+), and NH(4)(+) gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na(+) uptake and NH(3)/NH(4)(+) excretion were detected at most mitochondrion-rich cells (MRCs). H(+) probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 muM) blocked 35% of the NH(3)/NH(4)(+) secretion and 54% of the Na(+) uptake, suggesting that the Na(+)/H(+) exchanger (NHE) is involved in Na(+) and NH(3)/NH(4)(+) transport. Low-Na(+) water (<0.001 mM) or high-NH(4)(+) (5 mM) acclimation simultaneously increased Na(+) uptake and NH(3)/NH(4)(+) excretion but decreased or even reversed the H(+) gradient at the skin and MRCs. The correlation between NH(4)(+) production and H(+) consumption at the skin surface suggests that MRCs excrete nonionic NH(3) (base) by an acid-trapping mechanism. Raising the external NH(4)(+) significantly blocked NH(3)/NH(4)(+) excretion and Na(+) uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH(3)/NH(4)(+) excretion and Na(+) uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na(+)/H(+) exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na(+) acclimation. This study suggests a novel Na(+)/NH(4)(+) exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H(+) gradients across apical membranes of MRCs.

Exposure to copper nanoparticles impairs ion uptake, and acid and ammonia excretion by ionocytes in zebrafish embryos.

The potential toxicity of copper nanoparticles (CuNPs) to early stages of fishes is not fully understood, and little is known about their effects on ionocytes and associated functions. This study used zebrafish embryos as a model to investigate the toxic effects of CuNPs on two subtypes of ionocytes. Zebrafish embryos were exposed to 0.1, 1, and 3 mg L-1 CuNPs for 96 h. After exposure, whole-body Na+ and Ca2+ contents were significantly reduced at ≥0.1 mg L-1, while the K+ content had decreased at ≥1 mg L-1. H+ and NH4+ excretion by the skin significantly decreased at ≥1 mg L-1. The number of living ionocytes labeled with rhodamine-123 had significantly decreased with ≥0.1 mg L-1 CuNPs. The ionocyte subtypes of H+-ATPase-rich (HR) and Na+/K+-ATPase-rich (NaR) cells were labeled by immunostaining and had decreased with ≥1 mg L-1. Shrinkage of the apical opening of ionocytes was revealed by scanning electronic microscopy. Functional impairment was also reflected by changes in gene expressions, including ion transporters/channels and Ca2+-regulatory hormones. This study shows that CuNP exposure can impair two subtypes of ionocytes and their associated functions, including Na+/Ca2+ uptake and H+/NH4+ excretion in zebrafish embryos.

Specific expression and regulation of glucose transporters in zebrafish ionocytes.

Glucose, a carbohydrate metabolite, plays a major role in the energy supply for fish iono- and osmoregulation, and the way that glucose is transported in ionocytes is a critical process related to the functional operations of ionocytes. Eighteen members of glucose transporters (GLUTs, SLC2A) were cloned and identified from zebrafish. Previously, Na(+),K(+)-ATPase-rich (NaR), Na(+)-Cl(-) cotransporter-expressing (NCC), H(+)-ATPase-rich (HR), and glycogen-rich (GR) cells have been identified to be responsible for Ca(2+) uptake, Cl(-) uptake, Na(+) uptake, and the energy deposition, respectively, in zebrafish skin/gills. The purpose of the present study was to test the hypothesis of whether GLUT isoforms are specifically expressed and function in ionocytes to supply energy for ion regulatory mechanisms. On the basis of translational knockdown of foxi3a/3b (2 transcriptional factors related to the ionocytes' differentiation) and triple in situ hybridization/immunocytochemistry, 3 GLUT isoforms, zglut1a, -6, and -13.1, were specifically localized in NaR/NCC cells, GR cells, and HR cells, respectively. mRNA expression of zglut1a in embryos and adult gills were stimulated by the low Ca(2+) or low Cl(-) freshwater, which has been previously reported to upregulate the functions (monitored by epithelial Ca(2+) channel, NCC mRNA) of NaR/NCC cells, respectively while that of zglut13.1 was stimulated only by low Na(+), a situation to upregulate the function (monitored by carbonic anhydrase 15a mRNA) of HR cells. On the other hand, ambient ion compositions did not affect the zglut6 mRNA expression. Taken together, zGLUT1a, -6, and 13.1, the specific transporters in NaR/NCC cells, GR cells, and HR cells, may absorb glucose into the respective cells to fulfill different physiological demands.

Potassium Regulation in Medaka (Oryzias latipes) Larvae Acclimated to Fresh Water: Passive Uptake and Active Secretion by the Skin Cells.

Molecular mechanisms of Na+, Cl-, and Ca2+ regulation in ionocytes of fish have been well investigated. However, the regulatory mechanism of K+ in fishes has been largely unknown. In this study, we investigated the mechanism of K+ regulation in medaka larvae acclimated to fresh water. Using a scanning ion-selective electrode technique (SIET) to measure the K+ fluxes at skin cells, significant K+ effluxes were found at ionocytes; in contrast, significant K+ influxes were found at the boundaries between keratinocytes. High K+ water (HK) acclimation induced the K+ effluxes at ionocytes and suppressed the K+ influxes at keratinocytes. The K+ effluxes of ionocytes were suppressed by VU591, bumetanide and ouabain. The K+ influxes of keratinocytes were suppressed by TAP. In situ hybridization analysis showed that mRNA of ROMKa was expressed by ionocytes in the skin and gills of medaka larvae. Quantitative PCR showed that mRNA levels of ROMKa and NKCC1a in gills of adult medaka were upregulated after HK acclimation. This study suggests that medaka obtain K+ through a paracellular pathway between keratinocytes and extrude K+ through ionocytes; apical ROMKa and basolateral NKCC1a are involved in the K+ secretion by ionocytes.