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Behaviors are the integrative outcomes of the nervous system, which senses and responds to the internal physiological status and external stimuli. Teleosts are aquatic organisms which are more easily affected by the surrounding environment compared to terrestrial animals. To date, behavioral tests have been widely used to assess potential environmental risks using fish as model animals. In this review, we summarized recent studies regarding the effects of internal and external stimuli on fish behaviors. We concluded that behaviors reflect environmental and physiological changes, which have possible implications for environmental and physiological assessments.

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.

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.

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 (NH

Ionocytes in the skin and gills of seawater (SW) fishes are responsible for acid-base regulation and salt secretion. Na

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

Background Asian studies on soft tissue sarcoma (STS) incidence, irrespective of the primary site, are scant. Methods STS data were acquired from the population-based 2007–2013 Taiwan Cancer Registry of the Health and Welfare Data Science Center, Taiwan. Histological subtype-, site-, sex-, and age-specific STS incidence rates were analyzed according to the 2013 classification of the World Health Organization. Results In total, 11,393 patients with an age-standardized incidence rate (ASIR) of 5.62 (95% confidence interval, 5.51–5.73) per 100,000 person-years were identified. Overall, a male predominance (sex-standardized incidence rate ratio, 1.2) was noted, and the rate increased with age, peaking at >75 years. Approximately 30% of STSs occurred in connective, subcutaneous, and other soft tissues and 70% in other sites. In addition to connective, subcutaneous, and other soft tissues, the three most common primary sites were the stomach (15.9%), skin (14.3%), and small intestines (10.5%). Gastrointestinal stromal tumor was the most common subtype (29.2%; ASIR, 1.55/100,000 person-years), followed by liposarcoma (11.5%; ASIR, 0.63/100,000 person-years) and leiomyosarcoma (9.7%; ASIR, 0.53/100,000 person-years). Compared with relevant data from Western countries, the incidence rate of angiosarcomas was higher than that in other regions, whereas the incidence rates of leiomyosarcoma and Kaposi sarcoma were lower than those in other regions. Conclusion STS incidence varied by histological subtype, sex, age, and primary site in an Asian population. Our results suggested regional and racial discrepancies in the incidence rates of certain STS subtypes.

Pharmaceuticals and personal care products are emerging environmental pollutants. Cisplatin, one of the most widely used platinum-based chemotherapeutic agents, has been found to contaminate aquatic environments. Using zebrafish embryos as a model, cisplatin was previously found to impair skin ionocytes and ion regulation. The purpose of this study was to further investigate how cisplatin damages ionocytes. Zebrafish embryos were exposed to cisplatin (0, 50, and 100 μM) for 96 h (4-100 h post-fertilization) and then stained with fluorescent dyes to reveal mitochondrial activity (rhodamine123), apoptosis (acridine orange), and oxidative stress (CellROX/MitoSOX) in ionocytes of living embryos. Results showed that cisplatin exposure decreased rhodamine 123-labeled ionocytes, induced oxidative stress in ionocytes, and promoted apoptosis in a concentration-dependent manner. Quantitative PCR analysis showed that mRNA levels of antioxidative genes (sod1, sod2, gpx1a, and cat) and an apoptotic gene (caps3a) were induced. In the time-course experiment at 96-98 h post-fertilization, cisplatin increased oxidative stress and apoptosis in ionocytes in a time-dependent manner. In conclusion, this study demonstrates that cisplatin exposure induces oxidative stress, mitochondrial damage, and apoptosis in ionocytes of 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.

Environmental contamination by antibiotics has become a global issue. Colistin, a cationic antimicrobial polypeptide, has been widely used in human/veterinary medicine, and growth promotion in aquaculture. However, no study has been conducted to test the toxic effects of colistin on aquatic animals. In this study, we examined the effects of colistin on zebrafish embryos. Zebrafish embryos were incubated in different concentrations (0, 0.01, 0.1, 1, 2, 3, and 10 μM) of colistin for 96 h. Colistin increased the mortality rate in a dose-dependent manner (LC50 was 3.0 μM or 3.5 mg L−1), but it did not change the hatching rate, heart rate, body length, eye size, or yolk size of embryos. However, colistin impaired keratinocytes and lateral-line hair cells in the skin of embryos. Colistin (at concentrations ≥0.1 μM) decreased the number of FM1-43-labeled hair cells and reduced the mechanotransduction-mediated Ca2+ influx at hair bundles, suggesting that sublethal concentrations of colistin can impair lateral line function. To investigate the lethal injury, morphological changes were sequentially observed in post-hatched embryos subjected to lethal concentrations of colistin. We found that skin keratinocytes were severely damaged and detached after exposure, leading to hypotonic swelling of the yolk sac, loss of ion contents, cell lysis, and eventual death. This study revealed that acute colistin exposure can impair skin cells and pose a threat to fish survival.

Silver and silver nanoparticles are used in several consumer products, particularly sterilizing agents. Ag+ released from the particles causes physiological damages of aquatic organisms. However, the effects of silver on neural and behavioral functions of fish remain unclear. Here, we used zebrafish as a model to investigate the impacts of silver on social, learning and memory behaviors in teleost. Adult zebrafish showed mortality rates of 12.875% and 100% on 72 h exposure to 30 and ≥ 50 ppb of silver nitrate, respectively. Silver accumulation in the brain increased on exposure to 10 and 30 ppb of AgNO3. The physical fitness of the zebrafish, measured by novel tank diving test and swimming performance, decreased after 72 h incubation in 30 ppb of AgNO3. Exposure to 10 ppb of AgNO3 impaired social preference, social recognition, learning, and memory, but did not affect anxiety level, aggressiveness, and shoaling behavior. In situ hybridization of c-fos mRNA showed that AgNO3 treatment decreased neural activity in the brain areas crucial for learning, memory, and social behaviors, including the medial and dorsal zones of the dorsal telencephalic area. In conclusion, 72 h exposure to AgNO3 in a sublethal level impaired learning and social behaviors, indicating neurotoxicity in adult zebrafish.

Ammonia (including NH3 and NH4+) is a major pollutant of freshwater environments. However, the toxic effects of ammonia on the early stages of fish are not fully understood, and little is known about the effects on the sensory system. In this study, we hypothesized that ammonia exposure can cause adverse effects on embryonic development and impair the lateral line system of fish. Zebrafish embryos were exposed to high-ammonia water (10, 15, 20, 25, and 30 mM NH4Cl; pH 7.0) for 96 h (0–96 h post-fertilization). The body length, heart rate, and otic vesicle size had significantly decreased with ≥15 mM NH4Cl, while the number and function of lateral-line hair cells had decreased with ≥10 mM NH4Cl. The mechanoelectrical transduction (MET) channel-mediated Ca2+ influx was measured with a scanning ion-selective microelectrode technique to reveal the function of hair cells. We found that NH4+ (≥5 mM NH4Cl) entered hair cells and suppressed the Ca2+ influx of hair cells. Neomycin and La3+ (MET channel blockers) suppressed NH4+ influx, suggesting that NH4+ enters hair cells via MET channels in hair bundles. In conclusion, this study showed that ammonia exposure (≥10 mM NH4Cl) can cause adverse effects in zebrafish embryos, and lateral-line hair cells are sensitive to ammonia exposure.

Background The incidence of immune thrombocytopenia (ITP) is not well known in Asians. The aims of this study were to survey incidences and clinical features of ITP in Taiwan. Study design and methods This study identified 4855 incident ITP cases from the population-based National Health Insurance Research Database from mid-2006 to mid-2013, and compared incidences, patient characteristics, and clinical manifestations of ITP by age. Results Respective ITP incidence rates among those aged 15 years. Conclusion Incidence estimates of ITP in Taiwan were close to those of Western countries, with age-specific variations in sex ratio, comorbidity, splenectomy, secondary causes, and incidence trends. The results suggest no racial variations in ITP incidences, but a geographical difference in causes of secondary ITP.

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

This work was supported by the European Social Fund under the No. 09.3.3- LMT-K-712 “Development of Competences of Scientists, other Researchers and Students through Practical Research Activities” measure. AK would like to express sincere gratitude for Fellowship administrated by The Japan Society for the Promotion of Science (JSPS). Fellow’s ID No.: L12546. Authors are grateful to R. Vargalis (Vilnius University) for taking SEM images. © 2020. This work is licensed under a CC BY-NC-ND license., In the present work, Fe3+ and Zn2+ co-substituted β-tricalcium phosphate (β-TCP) has been synthesized by wet co-precipitation method. Co-substitution level in the range from 1 to 5 mol% has been studied. Thermal decomposition of as-prepared precipitates was shown to be affected by introducing of foreign ions, decreasing the decomposition temperature of precursor. It was determined that partial substitution of Ca2+ by Fe3+ and Zn2+ ions leads to the change in lattice parameters, which gradually decrease as doping level increases. Lattice distortion was also confirmed by means of Raman spectroscopy, which showed gradual change of the peaks shape in the Raman spectra. Rietveld refinement and electron paramagnetic resonance study confirmed that Fe3+ ions occupy only one Ca crystallographic site until Fe3+ and Zn2+ substitution level reaches 5 mol%. All co-substituted samples revealed paramagnetic behavior, magnetization of powders was determined to be linearly dependent on concentration of Fe3+ ions. Cytotoxicity of the synthesized species was estimated by in vivo assay using zebrafish (Danio rerio) and revealed non-toxic nature of the samples. Preparation of ceramic bodies from the powders was performed, however the results obtained on Vickers hardness of the ceramics did not show improvement in mechanical properties induced by co-substitution.----/ / /---- This is the preprint version of the following article: Lauryna Sinusaite, Anton Popov, Andris Antuzevics, Kestutis Mazeika, Dalis Baltrunas, Jen-Chang Yang, Jiun Lin Horng, Shengfang Shi, Tohru Sekino, Kunio Ishikawa, Aivaras Kareiva, Aleksej Zarkov, Fe and Zn co-substituted beta-tricalcium phosphate (β-TCP): Synthesis, structural, magnetic, mechanical and biological properties, Materials Science and Engineering: C 112, 110918 (2020), https://doi.org/10.1016/j.msec.2020.110918, which has been published in final form at https://www.sciencedirect.com/science/article/abs/pii/S0928493119344285#!. This article may be used for non-commercial purposes in accordance with Elsevier Terms and Conditions for Sharing and Self-Archiving. This work is licensed under a CC BY-NC-ND license., European Social Fund under the No. 09.3.3- LMT-K-712; Institute of Solid State Physics, University of Latvia as the Center of Excellence has received funding from the European Union’s Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017-TeamingPhase2 under grant agreement No. 739508, project CAMART².

The potential toxicity of nanoparticles (NPs) to the early stages of fish is still unclear. In this study, we investigated the toxic effects of silver (AgNPs) and copper nanoparticles (CuNPs) on lateral-line hair cells of zebrafish embryos. Zebrafish embryos were incubated in different concentrations of AgNPs and CuNPs at 0˜96 h post-fertilization (hpf). Both AgNPs and CuNPs were found to cause toxic effects in zebrafish embryos in a dose-dependent manner. Values of the 96-h 50% lethal concentration (LC50) of AgNPs and CuNPs were 6.1 ppm (56.5 μM) and 2.61 ppm (41.1 μM), respectively. The number of FM1-43-labeled hair cells and the microstructure of hair bundles were significantly impaired by AgNPs [≥1 ppm (9.3 μM)] and CuNPs [≥0.01 ppm (0.16 μM)]. Ca2+ influxes at hair bundles of hair cells were measured with a scanning ion-selective microelectrode technique to evaluate the function of hair cells. AgNPs [≥0.1 ppm (0.9 μM)] and CuNPs [≥0.01 ppm (0.16 μM)] were both found to significantly reduce Ca2+ influxes. Similar toxic effects were also found in hatched embryos subjected to 4 h of exposure (96˜100 hpf) to AgNPs and CuNPs. This study revealed that lateral-line hair cells of zebrafish are susceptible to AgNPs and CuNPs, and these contaminants in aquatic environments could pose a threat to fish survival.

Chitosan is suggested as no or low toxicity and biocompatible biomaterial. Digestion of chitosan to reduce molecular weight and formulate nanoparticle was generally used to improve efficiency for DNA or protein delivery. However, the toxicity of low-molecular-weight chitosan (LMWCS) towards freshwater fishes has not been well evaluated. Here, we reported the toxic mechanism of LMWCS using zebrafish (Danio rerio) liver (ZFL) cell line, zebrafish larvae, and adult fish. LMWCS rapidly induced cytotoxicity of ZFL cells and death of zebrafish. Cell membrane damaged by LMWCS reduced cell viability. Damaged membrane of epithelial cell in zebrafish larvae induced breakage of the yolk. Adult fish exhibited hypoxia before death due to multiple damages induced by LMWCS. Although the toxicity of LMWCS was revealed in zebrafish model, the toxicity was only present in pH

Acidification of freshwater ecosystems is recognized as a global environmental problem. However, the influence of acidic water on the early stages of freshwater fish is still unclear. This study focused on the sublethal effects of acidic water on the lateral line system of zebrafish embryos. Zebrafish embryos were exposed to water at different pH values (pH 4, 5, 7, 9, and 10) for 96 (0–96 h post-fertilization (hpf)) and 48 h (48∼96 hpf). The survival rate, body length, and heart rate significantly decreased in pH 4-exposed embryos during the 96-h incubation. The number of lateral-line neuromasts and the size of otic vesicles/otoliths also decreased in pH 4-exposed embryos subjected to 96- and 48-h incubations. The number of neuromasts decreased in pH 5-exposed embryos during the 96-h incubation. Alkaline water (pH 9 and 10) did not influence embryonic development but suppressed the hatching process. The mechanotransducer channel-mediated Ca2+ influx was measured to reveal the function of lateral line hair cells. The Ca2+ influx of hair cells decreased in pH 5-exposed embryos subjected to the 48-h incubation, and both the number and Ca2+ influx of hair cells had decreased in pH 5-exposed embryos after 96 h of incubation. In addition, the number and function of hair cells were suppressed in H+-ATPase- or GCM2-knockdown embryos, which partially lost the ability to secrete acid into the ambient water. In conclusion, this study suggests that lateral line hair cells are sensitive to an acidic environment, and freshwater acidification could be a threat to the early stages of fishes.

This study aimed to describe cancer incidence rates and trends specifically for adolescents aged 15-19 years during 1995-2009 in Taiwan. The incidence counts and census data were obtained from the population-based Taiwan Cancer Registry. During the 15-year study period, 4122 adolescents were diagnosed with cancer. The overall incidence rate was 155.2 per million person-years. Other epithelial tumors were the most frequently diagnosed cancer group (23.7%), followed by leukemias (18.0%) and lymphomas (13.9%). When compared to rates in Western countries, a significantly low rate of lymphomas was found. Moreover, rates of the subtypes of melanomas and nasopharyngeal carcinomas being 1/10- and 4-times rates in Western countries were the most striking variations. During 1995-2009, the overall rate of adolescent cancer did not significantly change. However, the most significant upward and declining trends in incidence rates were found for male germ cell neoplasms (annual percent change, APC, 6.4%) and hepatic tumors (APC, -11.1%), respectively. Further investigation and enhancement of the public discourse of possible lifestyle and environmental risk factors associated with increasing trends of certain adolescent cancers should be carried out in Taiwan.

Environmental contamination by anticancer pharmaceuticals has been widely reported. These drugs are not readily biodegradable, and their parent compounds and/or metabolites have been detected in surface waters and groundwater throughout the world. Adverse effects of anticancer drugs occur frequently in cancer patients, and a large body of clinical knowledge has accumulated. However, the effects of these drugs on aquatic organisms have not been thoroughly studied. This study aimed to investigate the effects of acute exposure to a common anticancer drug, vincristine (VCR), on zebrafish embryonic development and skin function. After 96 h of VCR exposure (0, 1, 10, 15, and 25 mg/L), significant teratogenic effects were observed, including growth retardation, pericardial edema, spine, tail, and yolk sac malformations (VCR ≥ 15 mg/L), a decreased heart rate, and ocular malformations (VCR ≥ 10 mg/L). The value of the half lethal concentration for zebrafish embryos was 20.6 mg/L. At ≥10 mg/L VCR, systemic ion contents and acid secretion in the skin over the yolk-sac decreased, and these findings were associated with decreases in skin ionocytes (H+-ATPase-rich cells and Na+-K+-ATPase-rich cells). Also, the microridge-structure of skin keratinocytes was significantly damaged. The number of lateral line hair cells was reduced when VCR was ≥10 mg/L, and functional impairment was detected when VCR was as low as 1 mg/L. Results of this in vivo study in zebrafish embryos indicate that acute exposure to VCR can lead to developmental defects, impairment of skin functions, and even fish death.

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.

Synovial sarcoma is a rare but highly malignant and metastatic disease. Despite its relative sensitivity to chemotherapies, the high recurrence and low 5-year survival rate for this disease suggest that new effective therapeutic agents are urgently needed. Marine antimicrobial peptide epinecidin-1 (epi-1), which was identified from orange-spotted grouper (Epinephelus coioides), exhibits multiple biological effects, including bactericidal, immunomodulatory, and anticancer activities. However, the cytotoxic effects and mechanisms of epi-1 on human synovial sarcoma cells are still unclear. In this study, we report that epi-1 exhibits prominent antisynovial sarcoma activity in vitro and in a human SW982 synovial sarcoma xenograft model. Furthermore, we determined that calcium overload-induced calpain activation and subsequent oxidative stress and mitochondrial dysfunction are required for epi-1-mediated cytotoxicity. Interestingly, reactive oxygen species (ROS)-mediated activation of extracellular signal-regulated kinase (ERK) plays a protective role against epi-1-induced cytotoxicity. Our results provide insight into the molecular mechanisms underlying epi-1-induced cell death in human SW982 cells.

This study aimed to assess the sublethal effects of a platinum-based compound, cisplatin, using a zebrafish model. Zebrafish embryos were incubated in different concentrations of cisplatin at 0–96 h post-fertilization. Using a non-invasive, scanning ion-selective electrode technique (SIET), we measured the functions of hair cells (Ca2+ influx) and ionocytes ([H+] gradients). The survival rate, hatching rate, phenotype, body length, whole-body ion (Na+, Cl−, and Ca2+) and Pt contents were also determined. The effects of cisplatin on zebrafish embryos were demonstrated as first impairing hair cell function (at 1 μM of cisplatin), the hair cell number, and body ion content of Cl− (at 10 μM of cisplatin), then decreasing ionocyte acid secretion and overall body ion contents of Na+ and Ca2+ (at 50 μM of cisplatin). The body length and ionocyte density decreased at 100 μM of cisplatin, and survival decreased at 500 μM of cisplatin. As the cisplatin concentration increased, the accumulation of Pt in fish embryos also increased. These results revealed that hair cells are significantly more susceptible to cisplatin toxicity than ionocytes. By determining the lowest observed effective concentration of cisplatin that caused in vivo functional alterations of zebrafish hair cells and skin ionocytes, this model demonstrated 500-fold greater sensitivity than by detecting changes in survival, for early assessment of the effects of platinum-based chemotherapeutic drugs on fish.

The calcium-sensing receptor (CaSR) is an extracellular Ca2+ sensor that plays a critical role in maintaining Ca2+ homeostasis in several organs, including the parathyroid gland and kidneys. In this study, through in situ hybridization, the expression of CaSR mRNA was found in the neuromasts of zebrafish larvae. Immunohistochemistry further demonstrated that the CaSR protein was present in neuromast hair cell stereocilia and basolateral membranes. Based on the expression and subcellular localization of the CaSR in hair cells, we hypothesized that the CaSR is expressed in zebrafish lateral-line hair cells to regulate mechanotransducer (MET)-channel-mediated Ca2+ entry. Using the scanning ion-selective electrode technique, MET-channel-mediated Ca2+ influx at the stereocilia of hair cells was measured in intact larvae. Ca2+ influx was suppressed after larvae were pretreated with a CaSR activator (R-568) or high-Ca2+ (HCa) medium. Gene knockdown by using morpholino oligonucleotides decreased CaSR expression in hair cells and eliminated the effects of R-568 and HCa on Ca2+ influx. In addition, we found that treatment with R-568 attenuated neomycin-induced hair cell death. This study is the first to demonstrate that the CaSR is involved in mechanotransduction in zebrafish hair cells.

A 2-year-old Asian girl presented to our facility for the evaluation of thrombocytopenia. She was treated with intravenous immunoglobulin under the impression of immune thrombocytopenia. However, her body temperature spiked and progressive pancytopenia, hepatosplenomegaly, abnormal liver function, coagulopathy, and pulmonary infiltration developed. The final diagnosis was systemic Epstein-Barr virus (EBV)-positive T-cell lymphoma of childhood with hemophagocytic syndrome. This type of cancer is extremely rare but occurs more commonly in Asians. Its prognosis is generally poor, and a treatment strategy is yet to be established. Double staining for EBV-encoded RNA and CD3 or CD8 is crucial for diagnosis. This type of lymphoma must be diagnosed differentially from acute EBV-associated hemophagocytic lymphohistiocytosis, which is considered nonmalignant. This case report highlights the importance of awareness of this type of rare cancer, a comprehensive diagnostic approach, and close communication between primary care physicians and pathologists.

Background & Aims This study examined and compared the incidence patterns of hepatocellular carcinoma among age groups in Taiwan, 30years after a universal hepatitis B virus immunization program was launched. Methods Data for hepatocellular carcinoma diagnosed in 2003–2011 were collected from the population-based Taiwan Cancer Registry. Age-standardized incidence rates were calculated to analyze and compare the changes in incidence rates and trends. More specific analyses were performed on four age groups separated by sex. Results A total of 82,856 patients were diagnosed with hepatocellular carcinoma in 2003–2011 in Taiwan, yielding an age-standardized incidence rate of 32.97 per 100,000 person-years. Hepatocellular carcinoma was predominantly diagnosed in middle-aged adults (50.1%) and elderly people (49.1%), in contrast to the low incidences in children (0.04%) and adolescents and young adults (0.8%). Striking variations in trends were found for children (annual percent change: −16.6%, 2003–2010) and adolescents and young adults (annual percent change: −7.9%, 2003–2011). The incidence rate of hepatocellular carcinoma in children decreased to zero in 2011; only a slight decline in trends occurred for the middle-aged group (annual percent change: −2%, 2003–2011), and a slight upward trend was observed for elderly people (1.3%), specifically in women (1.7%). Conclusions In Taiwan, hepatitis B virus-related hepatocellular carcinoma was nearly eradicated in children in 2011. The findings on age-specific incidence patterns and trends of hepatocellular carcinoma suggest that different control strategies for treating this devastating disease in the future be made according to age.

Summary Evidence from our previous study suggested that the incidence of germ cell neoplasms in children and adolescents is increasing. The objectives of this analysis were to quantify this trend in patients aged 0–9 and 10–19 years (pre-pubertal and adolescent groups, respectively) and compare rates in Taiwan according to geographic distribution. Germ cell neoplasm frequencies among 1267 patients aged 0–19 years spanning 1995–2009 were obtained from the population-based Taiwan Cancer Registry. The incidence patterns according to sex, age, disease subgroup, and geographic distribution were analyzed. The incidence rates in the pre-pubertal and adolescent groups were 10.58 and 16.06 per million person-years, respectively. The overall rates increased significantly by 3.2% annually in the adolescent group during the 15-year study period, and increased only among the males. In contrast, no change in trend was observed in the pre-pubertal group. Subgroup analysis showed significant upward trends in the incidence rates of intracranial/intraspinal and testicular germ cell tumors (GCTs) in the adolescent males and extracranial/extragonadal GCTs in the pre-pubertal boys. The most striking differences between the study population and white Americans were that the rates of testicular GCTs were 5-fold higher and 4-fold lower in the Taiwanese pre-pubertal and adolescent groups, respectively. Significantly higher rates were found in Hualien and Chiayi Counties compared with the other areas of Taiwan. The upward trend of testicular GCTs in the adolescent males is consistent with findings from Western countries. The underlying causes that led to the high rate of testicular GCTs in the pre-pubertal boys and significantly higher rates in specific counties warrant further investigation.

The incidence of immune thrombocytopenia (ITP) is not well known in Asians. The aims of this study were to survey incidences and clinical features of ITP in Taiwan.This study identified 4855 incident ITP cases from the population-based National Health Insurance Research Database from mid-2006 to mid-2013, and compared incidences, patient characteristics, and clinical manifestations of ITP by age.Respective ITP incidence rates among those aged15, 15 to 59, and ≥60 years were 4.0, 2.0, and 5.4 per 100,000 person-years. A male predominance was noted in children, and a female predominance was found in adults. The most common causes of secondary ITP were systemic lupus erythematosus (21.8%), viral hepatitis C (16.9%), and viral hepatitis B (13.4%). The rate of secondary ITP in children was less than one fifth that in adults (4.2% vs. 23.8%). Rates of central nervous system (1.1%) and gastrointestinal tract bleeding (3.3%) were rare, with variations by age. The rate of splenectomies in children (0.4%) was only one tenth that in adults (4.1%). The disease in 25% of children and 30% of adults became persistent or chronic. A decreasing trend in the ITP incidence was found (annual percentage change, -4.9%), and it was confined to those aged15 years.Incidence estimates of ITP in Taiwan were close to those of Western countries, with age-specific variations in sex ratio, comorbidity, splenectomy, secondary causes, and incidence trends. The results suggest no racial variations in ITP incidences, but a geographical difference in causes of secondary ITP.

BACKGROUND Currently, little information is available on childhood cancer incidence rates in Eastern Asia. The objective of this study was to report the first population-based cancer surveillance of children and adolescents in Taiwan. METHODS Data from the Taiwan Cancer Registry were examined for cancer frequencies and incidence rates among individuals ages birth to 19 years from 1995 to 2009. Types of cancers were grouped according to the International Classification of Childhood Cancer. Rates were compared by sex and age. For further comparisons with other countries, rates were age standardized to the 2000 world standard population in 5-year age groups. Trends in incidence rates also were evaluated. RESULTS In total, 12,315 individuals were diagnosed with childhood cancers, for an age-standardized incidence rate (ASR) of 132.1 per million person-years from 1995 to 2009. The male-to-female incidence rate ratio was 1.19. Overall, leukemias were the most common cancer (ASR, 39.1 per million person-years), followed by central nervous system neoplasms (15.8 per million person-years), and lymphomas (15.3 per million person-years). During the 15-year study period, the incidence rates increased by 1% annually. Compared with other countries, the rate of hepatic tumors was 2 times greater in Taiwan. The rate of germ cell neoplasms in Taiwan was similar to that in the United States and was 1.3 to 1.9 times greater compared with Canada, Brazil, Israel, and Japan. CONCLUSIONS Based on the current data, the observed increase in overall incidence rates was attributable only marginally to improvements in case ascertainment and diagnostic procedures. The high rates of malignant hepatic tumors and germ cell neoplasms in Taiwan suggest variations in the background risk factors. Cancer 2014;120:3545–3553. © 2014 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. The authors examine cancer incidence patterns in children and adolescents based on a national, population-based cancer registry of 12,315 individuals in Taiwan from 1995 to 2009. The high rates of malignant hepatic tumors and germ cell neoplasms in Taiwan suggest variations in background risk factors.

Zebrafish lateral-line hair cells are an in vivo model for studying hair cell development, function, and ototoxicity. However, the molecular identification and properties of the mechanotransducer (MET) channel in hair cells are still controversial. In this study, a noninvasive electrophysiological method, the scanning ion-electrode technique (SIET), was applied for the first time to investigate properties of MET channels in intact zebrafish embryos. With the use of a Ca2+-selective microelectrode to deflect hair bundles and simultaneously record the Ca2+flux, the inward Ca2+flux was detected at stereocilia of hair cells in 2- to ∼4-day postfertilization embryos. Ca2+influx was blocked by MET channel blockers (BAPTA, La3+, Gd3+, and curare). In addition, 10 μM aminoglycoside antibiotics (neomycin and gentamicin) were found to effectively block Ca2+influx within 10 min. Elevating the external Ca2+level (0.2–2 mM) neutralized the effects of neomycin and gentamicin. However, elevating the Mg2+level up to 5 mM neutralized blockade by gentamicin but not by neomycin. This study demonstrated MET channel-mediated Ca2+entry at hair cells and showed that the SIET to be a sensitive approach for functionally assaying MET channels in zebrafish.

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.

Ionocytes in the skin and gills of seawater (SW) teleosts are responsible for both salt and acid secretion. However, the mechanism through which ionocytes secrete acid is still unclear. Here, we hypothesized that apical Na+/H+ exchangers (NHE2/3), carbonic anhydrase (CA2-like) and basolateral HCO3−/Cl− exchanger (AE1) are involved in acid secretion. In addition, the hypothesized involvement of basolateral AE1 suggested that acid secretion may be linked to Cl− secretion by ionocytes. The scanning ion-selective electrode technique (SIET) was used to measure H+ and Cl− secretion by ionocytes in the skin of medaka larvae acclimated to SW. Treatment with inhibitors of NHE, CA and AE suppressed both H+ and Cl− secretion by ionocytes. Short-term exposure to hypercapnic SW stimulated both H+ and Cl− secretion. mRNA of CA2-like and AE1 were localized to ionocytes in the skin. Branchial mRNA levels of NKCC1a, CA2-like and AE1a increased together with the salinity to which fish were acclimated. In addition, both AE1a and AE1b mRNA increased in fish acclimated to acidified (pH 7) SW; NKCC1a mRNA increased in fish acclimated to pH 9 SW. This study reveals the mechanism of H+ secretion by ionocytes and refines our understanding of the well-established mechanism of Cl− secretion by ionocytes of SW fish.

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.

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.

Population-based studies on soft tissue sarcomas (STS) incidence beyond extremities and trunk wall are lacking in Asia. In this study, we aimed to report the incidences of STS arise in any part of the body. The data of primary STS registered in the population-based Taiwan Cancer Registry during 2007 and 2013 was acquired via the Health and Welfare Data Science Center, Taiwan. Histologic subtype-, age-, and gender-specific STS incidence rates of all sites were analyzed. A total of 11393 subjects were identified, with an age-standardized incidence rate of 5.62 (95% CI, 5.51-5.73) per 100,000 person-years. Approximately 30% of STS occurred in soft tissues (ICD-O-3 topography codes of C47, C49), and 70% occurred in all other primary sites (ICD-O-3 topography codes of C00-C80 except C47, C49, C40 and C41). The three most common primary sites other than soft tissues were stomach (15.9%), skin (14.3%) and small intestine (10.5%). Gastrointestinal stromal tumor (GIST) was the most common subtype, accounting for 29.2% of STS, followed by liposarcoma (11.5%) and leiomyosarcoma (9.7%). For overall STS, no significant incidence trend was identified during 2007-2013. However, a significant declining trend in incidence rate of undifferentiated pleomorphic sarcoma was found (annual percentage change, APC, -7.8%). By contrast, a significant upward trend was identified in other specified sarcomas (APC, 7.3%). For the first time, this analysis demonstrated that STS incidence varied by histologic subtype, gender, age, and primary site in Asians. In the future, more population-based studies should be performed in Asians for comparison with the results from Western countries to better clarification if racial variation exists in any subtype-specific incidence rates of STS. Citation Format: Giun-Yi Hung, Jiun-Lin Horng, Chueh-Chuan Yen. Incidences of malignant primary soft-tissue sarcomas regardless of primary site: A nationwide population-based study in Taiwan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1204.

A noninvasive scanning ion-selective electrode technique (SIET) was applied to measure Cl−transport at individual mitochondrion-rich cells (MRCs) in the skin of euryhaline tilapia ( Oreochromis mossambicus) larvae. In seawater (SW)-acclimated larvae, outward Cl−gradients (20∼80 mM higher than the background) were measured at the surface, indicating a secretion of Cl−from the skin. By serial probing over the surface of MRCs and adjacent keratinocytes (KCs), a significant outward flux of Cl−was detected at the apical opening (membrane) of MRCs. Treatment with 100 μM ouabain or bumetanide inhibited the Cl−secretion by ∼75%. In freshwater (FW)-acclimated larvae, a lower level of outward Cl−gradients (0.2∼1 mM) was measured at the skin surface. Low-Cl−water (+-Cl−cotransporter (NCC) immunoreactivity of MRCs in the larval skin. An inward flux of Cl−was detected when probing the exterior surface of a group of MRCs (convex-MRCs) that express the NCC. An NCC inhibitor (100 μM metolazone) reduced the flux by ∼90%. This study provides direct and convincing evidence for Cl−transport by MRCs of SW- and FW-acclimated euryhaline tilapia and the involvement of an apical NCC in Cl−uptake of MRCs of FW-acclimated fish.

It is important to maintain internal pH homeostasis in biological systems. In our previous studies, H+-ATPase-rich (HR) cells were found to be responsible for proton secretion in the skin of zebrafish embryos during development. In this study, zebrafish embryos were exposed to acidic and basic waters to investigate the regulation of HR cell acid secretion during pH disturbances. Our results showed that the function of HR cells on the skin of zebrafish embryos can be upregulated in pH 4 water not only by increasing the cell number but also by enlarging the acid-secreting function of single cells. We also identified an “alveolar-type” apical opening under scanning electron microscopy observations of the apical membrane of HR cells, and the density and size of the alveolar type of apical openings were also increased in pH 4 water. p63 and PCNA immunostaining results also showed that additional HR cells in pH 4 water may be differentiated not only from ionocyte precursor cells but also newly proliferating epithelial stem cells.

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H+and NH4+fluxes in specific cells on the skin of zebrafish larvae. NH4+extrusion was relatively high in H+pump-rich cells, which were identified as the H+-secreting ionocyte in zebrafish. Minor NH4+extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH4+extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H+and NH4+gradients at the larval surface. Moreover, coupled decreases in NH4+and H+extrusion were found in larvae treated with an H+-pump inhibitor (bafilomycin A1) or H+-pump gene ( atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH4+excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H+pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.

H+-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na+uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H+activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H+activity and increased Na+uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H+-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na+fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na+uptake and acid-base regulation mechanisms in zebrafish HR cells.

Most epidemiological studies of soft tissue sarcoma (STS) were performed in the Western countries, and only limited data highlighting that in the Asian population. The aim of this study is to conduct a comprehensive analysis for the incidence rates of STS in Taiwan. This was a population-based study analyzing the incidence rates and trends of the primary STS over extremities and trunk wall during 2003 to 2011 by using the nationwide Taiwan Cancer Registry. More specific analyses were conducted for subtypes. Incidence rates of overall STS by cities and counties were also investigated. A total of 3843 cases were diagnosed with STS during the study period, giving an age-standardized rate (ASR) of 1.63 per 100,000 person-years. Liposarcoma was the most frequent subtype, followed by undifferentiated pleomorphic sarcoma and leiomyosarcoma. STS was more frequently diagnosed in males and angiosarcoma was the most prominent sex-specific type. ASR increased with age in most of the STS subtypes and varied by histologic subtype. The incidence of peripheral primitive neuroectodermal tumor was highest in children, whereas rhabdomyosarcoma revealed a bimodal age distribution. Annual percent change (APC) of STS was 2.2%, and significant change in trend was only in males (APC, 3.5%, P

The sensory hair cells in mammalian inner ear are responsible for sound transduction.Sound waves deflect hair bundles and open the mechanotransducer (MET) channels, which initiated mechanotransduct...

Background Evidence from our recent study suggested that the overall trend for cancer incidence in children and adolescents has been increasing in Taiwan. Methods To analyze geographic variations in this trend, cancer frequencies and incidence rates of disease groups were quantified according to geographic areas among 12,633 patients aged

The mammalian kidney excretes its metabolic acid load through the proton-transporting cells, intercalated cells, in the distal nephron and collecting duct. Fish excrete acid through external organs, gill, or skin; however, the cellular function is still controversial. In this study, molecular and electrophysiological approaches were used to identify a novel cell type secreting acid in skin of zebrafish ( Danio rerio) larvae. Among keratinocytes covering the larval surface, novel proton-secreting ionocytes, proton pump (H+-ATPase)-rich cells, were identified to generate strong outward H+flux. The present work demonstrates for the first time, with a noninvasive technique, H+-secreting cells in an intact animal model, the zebrafish, showing it to be a suitable model in which to study the functions of vertebrate transporting epithelia in vivo.

The perception of equilibrium and sound in fish depends on the deflection of hair bundles of hair cell by the otolith. However, the accreting nature of teleostean otoliths poses a problem for maintenance of proper contact between the hair bundle and the otolith surface. Immunocytochemical staining localizes abundant proton-secreting H-ATPase in the apical membrane of the hair cells. The H-ATPase-mediated proton secretion into the endolymph causes an approximately 0.4-unit pH decrease, which was quantified by an H-selective microelectrode. Thus, the hair cells maintain the proper distance from the otolith by neutralizing the alkaline endolymph to retard CaCO3 deposition on the otolith opposite the sensory macula. Carbonic anhydrase, which hydrolyses CO2 and produces HCO3 – and H, was also localized in the hair cells. Ionocytes showed prominent immunostaining of carbonic anhydrase and Na-K-ATPase, indicating its role in transepithelial transport of HCO3 – across the membranous labyrinth into the endolymph. Ionocytes form a ring closely surrounding the sensory macula. HCO3 – secreted from the ionocytes may serve as a barrier to neutralize H diffused from the sensory macula while keeping the endolymph alkaline outside the sensory macula. The ingenious arrangement of ionocytes and hair cells results in a unique sculptured groove, which is a common feature on the proximal surface of all teleostean otoliths. J. Comp. Neurol. 488:331–341, 2005. © 2005 Wiley-Liss, Inc.

The anion exchanger (AE) plays critical roles in physiological processes including CO2 transport and volume regulation in erythrocytes and acid-base regulation in renal tubules. Although expression of the AE in inner-ear hair cells was reported, its specific localization and function are still unclear. Using in situ hybridization, we found that the AE1b transcript is expressed in lateral-line hair cells of zebrafish larvae. An immunohistochemical analysis with a zebrafish-specific antibody localized AE1b to stereocilia of hair cells, and the expression was eliminated by morpholino knockdown of AE1b. A non-invasive, scanning ion-selective electrode technique was applied to analyze mechanotransducer (MET) channel-mediated Ca2+ influx at stereocilia of hair cells of intact fish. Ca2+ influx was effectively suppressed by AE1b morpholino knockdown and inhibitor (DIDS) treatment. Elevating external Ca2+ (0.2 to 2 mM) neutralized the inhibition of DIDS. Taken together, this study provides solid evidence to show that AE1b in stereocilia is required for the proper functioning of MET channels.

Mammalian aquaporin 1 (AQP1) is well known to function as a membrane channel for H2O and CO2 transport. Zebrafish AQP1a.1 (the homologue of mammalian AQP1) was recently identified in ionocytes of embryos; however its role in ionocytes is still unclear. In this study, we hypothesized that zebrafish AQP1a.1 is involved in the acid secretion by ionocytes through facilitating H2O and CO2 diffusion. A real-time PCR showed that mRNA levels of AQP1a.1 in embryos were induced by exposure to 1% CO2 hypercapnia for 3 days. In situ hybridization and immunohistochemistry showed that the AQP1a.1 transcript was highly expressed by acid-secreting ionocytes, i.e., H+-ATPase-rich (HR) cells. A scanning ion-selective electrode technique (SIET) was applied to analyze CO2-induced H+ secretion by individual ionocytes in embryos. H+ secretion by HR cells remarkably increased after a transient loading of CO2 (1% for 10 min). AQP1a.1 knockdown with morpholino oligonucleotides decreased the H+ secretion of HR cells by about half and limited the CO2 stimulated increase. In addition, exposure to an AQP inhibitor (PCMB) for 10 min also suppressed CO2-induced H+ secretion. Results from this study support our hypothesis and provide in vivo evidence of the physiological role of AQP1 in CO2 transport.

In zebrafish, Rhcg1 was found in apical membranes of skin ionocytes [H+-ATPase-rich (HR) cells], which are similar to α-type intercalated cells in mammalian collecting ducts. However, the cellular distribution and role of Rhbg in zebrafish larvae have not been well investigated. In addition, HR cells were hypothesized to excrete ammonia against concentration gradients. In this study, we attempted to compare the roles of Rhbg and Rhcg1 in ammonia excretion by larval skin and compare the capability of skin cells to excrete ammonia against concentration gradients. Using in situ hybridization and immunohistochemistry, Rhbg was localized to both apical and basolateral membranes of skin keratinocytes. A scanning ion-selective electrode technique (SIET) was applied to measure the NH4+ flux at the apical surface of keratinocytes and HR cells. Knockdown of Rhbg with morpholino oligonucleotides suppressed ammonia excretion by keratinocytes and induced compensatory ammonia excretion by HR cells. To compare the capability of cells to excrete ammonia against gradients, NH4+ flux of cells was determined in larvae exposed to serial concentrations of external NH4+. Results showed that HR cells excreted NH4+ against higher NH4+ concentration than did keratinocytes. Knockdown of the expression of either Rhcg1 or H+-ATPase in HR cells suppressed the capability of HR cells.

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 ...