Cellular mechanisms underlying extraordinary sulfide tolerance in a crustacean holobiont from hydrothermal vents.

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulfide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulfide-rich vent systems. Despite the toxicity of hydrogen sulfide, X. testudinatus occupies an ecological niche in a sulfide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulfide detoxification. Here sulfide is oxidized to thiosulfate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulfate and hypotaurine is mediated by the sodium-independent sulfate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulfide-oxidizing endo-symbiotic bacteria, creating an effective sulfide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet.

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.

Systemic Epstein-Barr Virus-positive T-Cell Lymphoma of Childhood Presentation With Hemophagocytosis.

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.

Incidence of immune thrombocytopenia in Taiwan: a nationwide population-based study.

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, 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 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.

Changing incidence patterns of hepatocellular carcinoma among age groups in Taiwan.

BACKGROUND & AIMS: This study examined and compared the incidence patterns of hepatocellular carcinoma among age groups in Taiwan, 30 years 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.

A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes).

The ion regulation mechanisms of fishes have been recently studied in zebrafish (Danio rerio), a stenohaline species. However, recent advances using this organism are not necessarily applicable to euryhaline fishes. The euryhaline species medaka (Oryzias latipes), which, like zebrafish, is genetically well categorized and amenable to molecular manipulation, was proposed as an alternative model for studying osmoregulation during acclimation to different salinities. To establish its suitability as an alternative, the present study was conducted to (1) identify different types of ionocytes in the embryonic skin and (2) analyze gene expressions of the transporters during seawater acclimation. Double/triple in situ hybridization and/or immunocytochemistry revealed that freshwater (FW) medaka contain three types of ionocyte: (1) Na(+)/H(+) exchanger 3 (NHE3) cells with apical NHE3 and basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC), Na(+)-K(+)-ATPase (NKA) and anion exchanger (AE); (2) Na(+)-Cl(-) cotransporter (NCC) cells with apical NCC and basolateral H(+)-ATPase; and (3) epithelial Ca(2+) channel (ECaC) cells [presumed accessory (AC) cells] with apical ECaC. On the other hand, seawater (SW) medaka has a single predominant ionocyte type, which possesses apical cystic fibrosis transmembrane conductance regulator (CFTR) and NHE3 and basolateral NKCC and NKA and is accompanied by smaller AC cells that express lower levels of basolateral NKA. Reciprocal gene expressions of decreased NHE3, AE, NCC and ECaC and increased CFTR and NKCC in medaka gills during SW were revealed by quantative PCR analysis.

Acidified water promotes silver-induced toxicity in zebrafish embryos.

Freshwater acidification is a global environmental challenge, yet the effects of acidic water on fish resistance to toxic Ag+ remain an unexplored area. To address this knowledge gap, zebrafish embryos were exposed to different concentrations (0 (control), 0.1, and 0.25 mg/L) of AgNO3 under pH 5 or 7 for 7 days. Notably, AgNO3 at 0.25 mg/L resulted in 100 % mortality in both pH conditions, while AgNO3 at 0.1 mg/L resulted in higher mortality at pH 5 (85 %) compared to pH 7 (20 %), indicating that acidic water enhanced Ag+ toxicity. Several parameters, including body length, inner ear (otic vesicle and otolith) and yolk sac areas, lateral line hair cell number and morphology, the number of ionocytes (H+-ATP-rich cells and Na+/K+-ATP-rich cells), and ion contents (Ag+, Na+, and Ca2+) were assessed at 96 h (day 4) to investigate individual and combined effects of Ag+ and acid on embryos. Acid alone did not significantly alter most parameters, but it decreased the yolk sac area and increased the ionocyte number. Conversely, Ag+ alone caused reductions in most parameters, including body length, the inner ear area, hair cell number, and ionocyte number. Combining acid and Ag+ resulted in greater suppression of the otolith area, hair cell number, and Na+/Ca2+ contents. In conclusion, acidification of freshwater poses a potential risk to fish embryo viability by increasing their susceptibility to silver toxicity, specifically affecting sensory function and ion regulation.

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.

Assessing cardiovascular toxicity in zebrafish embryos exposed to copper nanoparticles.

The toxicity of copper nanoparticles (CuNPs) to aquatic animals, particularly their effects on the cardiovascular system, has not been thoroughly investigated. In the present study, zebrafish embryos were used as a model to address this issue. After exposure to different concentrations (0.01, 0.1, 1, and 3 mg/L) of CuNPs for 96 h (4 to 100 h post-fertilization), cardiac parameters of the heart rate (HR), end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), ejection fraction (EF), and cardiac output (CO), and vascular parameters of the aortic blood flow velocity (ABFV) and aortic diameter (AD) were examined by a video-microscopic method. Morphologically, CuNPs induced concentration-dependent pericardial edema. Although CuNPs did not alter the HR, they significantly reduced the EDV, SV, and CO at ≥0.1 mg/L, the ESV and EF at 3 mg/L, the ABFV at ≥0.1 mg/L, and the AD at ≥1 mg/L. Transcript levels of several cardiac genes, nppa, nppb, vmhc, and gata4, were also examined. CuNPs significantly suppressed nppa and nppb at ≥0.1 mg/L, gata4 at ≥0.01 mg/L, and vmhc at 1 mg/L. This study demonstrated that CuNPs can induce cardiovascular toxicity at environmentally relevant concentrations during fish embryonic development and highlight the potential ecotoxicity of CuNPs to aquatic animals.

Extracellular Ca(2+) and Mg(2+) modulate aminoglycoside blockade of mechanotransducer channel-mediated Ca(2+) entry in zebrafish hair cells: an in vivo study with the SIET.

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 Ca(2+)-selective microelectrode to deflect hair bundles and simultaneously record the Ca(2+) flux, the inward Ca(2+) flux was detected at stereocilia of hair cells in 2- to ~4-day postfertilization embryos. Ca(2+) influx was blocked by MET channel blockers (BAPTA, La(3+), Gd(3+), and curare). In addition, 10 µM aminoglycoside antibiotics (neomycin and gentamicin) were found to effectively block Ca(2+) influx within 10 min. Elevating the external Ca(2+) level (0.2-2 mM) neutralized the effects of neomycin and gentamicin. However, elevating the Mg(2+) level up to 5 mM neutralized blockade by gentamicin but not by neomycin. This study demonstrated MET channel-mediated Ca(2+) entry at hair cells and showed that the SIET to be a sensitive approach for functionally assaying MET channels in zebrafish.

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.

Rhcg1 and Rhbg mediate ammonia excretion by ionocytes and keratinocytes in the skin of zebrafish larvae: H+-ATPase-linked active ammonia excretion by ionocytes.

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.

Development in a naturally acidified environment: Na+/H+-exchanger 3-based proton secretion leads to CO2 tolerance in cephalopod embryos.

BACKGROUND: Regulation of pH homeostasis is a central feature of all animals to cope with acid-base disturbances caused by respiratory CO2. Although a large body of knowledge is available for vertebrate and mammalian pH regulatory systems, the mechanisms of pH regulation in marine invertebrates remain largely unexplored. RESULTS: We used squid (Sepioteuthis lessoniana), which are known as powerful acid-base regulators to investigate the pH regulatory machinery with a special focus on proton secretion pathways during environmental hypercapnia. We cloned a Rhesus protein (slRhP), V-type H+-ATPase (slVHA) and the Na+/H+ exchanger 3 (slNHE3) from S. lessoniana, which are hypothesized to represent key players in proton secretion pathways among different animal taxa. Specifically designed antibodies for S. lessoniana demonstrated the sub-cellular localization of NKA, VHA (basolateral) and NHE3 (apical) in epidermal ionocytes of early life stages. Gene expression analyses demonstrated that slNHE3, slVHA and slRhP are up regulated in response to environmental hypercapnia (pH 7.31; 0.46 kPa pCO2) in body and yolk tissues compared to control conditions (pH 8.1; 0.045 kPa pCO2). This observation is supported by H+ selective electrode measurements, which detected increased proton gradients in CO2 treated embryos. This compensatory proton secretion is EIPA sensitive and thus confirms the central role of NHE based proton secretion in cephalopods. CONCLUSION: The present work shows that in convergence to teleosts and mammalian pH regulatory systems, cephalopod early life stages have evolved a unique acid-base regulatory machinery located in epidermal ionocytes. Using cephalopod molluscs as an invertebrate model this work provides important insights regarding the unifying evolutionary principles of pH regulation in different animal taxa that enables them to cope with CO2 induced acid-base disturbances.

CO(2)-driven seawater acidification differentially affects development and molecular plasticity along life history of fish (Oryzias latipes).

Fish early life stages have been shown to react sensitive to simulated ocean acidification. In particular, acid-base disturbances elicited by altered seawater carbonate chemistry have been shown to induce pathologies in larval fish. However, the mechanisms underlying these disturbances are largely unknown. We used gene expression profiling of genes involved in acid-base regulation and metabolism to investigate the effects of seawater hypercapnia on developing Japanese ricefish (medaka; Oryzias latipes). Our results demonstrate that embryos respond with delayed development during the time window of 2-5 dpf when exposed to a seawater pCO(2) of 0.12 and 0.42 kPa. This developmental delay is associated with strong down-regulation of genes from major metabolic pathways including glycolysis (G6PDH), Krebs cycle (CS) and the electron transport chain (CytC). In a second step we identified acid-base relevant genes in different ontogenetic stages (embryos, hatchlings and adults) and tissues (gill and intestine) that are up regulated in response to hypercapnia, including NHE3, NBCa, NBCb, AE1a, AE1b, ATP1a1a.1, ATP1a1b, ATP1b1a, Rhag, Rhbg and Rhcg. Interestingly, NHE3 and Rhcg expressions were increased in response to environmental hypercapnia in all ontogenetic stages and tissues tested, indicating the central role of these proteins in acid-base regulation. Furthermore, the increased expression of genes from amino acid metabolism pathways (ALT1, ALT2, AST1a, AST1b, AST2 and GLUD) suggests that energetic demands of hatchlings are fueled by the breakdown of amino acids. The present study provides a first detailed gene expression analysis throughout the ontogeny of a euryhaline teleost in response to seawater hypercapnia, indicating highest sensitivity in early embryonic stages, when functional ion regulatory epithelia are not yet developed.

Toxic effects of polystyrene nanoparticles on the development, escape locomotion, and lateral-line sensory function of zebrafish embryos.

Environmental pollution by micro- and nanosized plastic particles is a potential threat to aquatic animals. Polystyrene is one of the most common plastic particles in aquatic environments. Previous studies found that polystyrene nanoparticles (PNs) can penetrate the integument and accumulate in the organs of fish embryos. However, the potential impacts of PNs on fish embryos are not fully understood. To investigate this issue, zebrafish embryos were exposed to different concentrations (10, 25, and 50 mg/L) of PNs (25 nm) for 96 h (4-100 h post-fertilization), and various endpoints were examined, including developmental morphology (body length, sizes of the eyes, otic vesicles, otoliths, pericardial cavity, and yolk sac), locomotion (touch-evoked escape response and spinal motor neurons), and lateral-line function (hair cell number and hair bundle number). Exposure to 50 mg/L of PNs resulted in significant adverse effects across all endpoints studied, indicating that embryonic development was severely disrupted, and both locomotion and sensory function were impaired. However, at 25 mg/L of PNs, only locomotion and sensory function were significantly affected. The effects were insignificant in all examined endpoints at 10 mg/L of PNs. Transcript levels of several marker genes for neuronal function and eye development were suppressed after treatment. Exposure to fluorescent PNs showed that they accumulated in various organs including, the eyes, gills, blood vessels, gallbladder, gut, and lateral line neuromasts. Overall, this study suggests that short-term exposure to a high concentration of PNs can threaten fish survival by impairing embryonic development, locomotion performance, and mechanical sensory function.

Exposure to silver impairs the osmoregulatory capability of euryhaline medaka (Oryzias latipes) subjected to salinity changes.

The widespread use of silver in nanomaterials has led to increases in environmental contamination, which poses a threat to aquatic animals. Euryhaline fish, which live in environments with fluctuating salinity levels, have strong osmotic regulatory abilities to cope with such changes. This study attempted to investigate how silver affects the osmoregulatory capabilities of euryhaline fish, using medaka (Oryzias latipes) embryos as a model. The embryos were exposed to AgNO3 for 7 d in either fresh water (FW) or seawater (SW), and their mortality, heart rate, morphology, and ionocytes were examined. Results showed that the toxicity of AgNO3 was higher in FW than in SW (50% lethal concentrations (LC50) were 0.17 vs. 1.01 ppm). Although AgNO3 (0.05 and 0.1 ppm) did not significantly change the morphology of embryos, it impaired ionocytes and elevated heart rates in FW. While, AgNO3 (0.1 and 0.5 ppm) did not affect the morphology, ionocytes, or heart rate in SW, it impaired the hypo-osmoregulatory capability and elevated the mortality of embryos that were transferred from FW to SW. At 12 h after SW transfer, ionocytes were severely impaired, and water-drinking behavior was suppressed, resulting in body dehydration and sodium overload. In contrast, AgNO3 did not elevate the mortality of embryos that were transferred from SW to FW. To sum up, the presence of silver in FW during the developmental stage of euryhaline fish could potentially endanger their survival during SW adaptation.

Sublethal effects of methylmercury on lateral line sensory and ion-regulatory functions in zebrafish embryos.

Methylmercury can interfere with the normal functioning of the nervous system, causing a variety of behavioral and physiological changes in fish. However, the influence of MeHg on the lateral line sensory and ion-regulatory functions of fish is not clear. Zebrafish embryos were utilized as a model to address this question. After exposure to water-borne MeHg (5, 10, 50, or 100 ppb) for 96 h (4-100 h post-fertilization), the survival rate declined by ca. 50 % at 100 ppb. However, MeHg at sublethal concentrations delayed hatching and decreased heart rates and body length. As to effects on the lateral line sensory system, MeHg at ≥10 ppb decreased the number of hair cells and impaired hair bundles and Ca2+-mediated mechanical transduction. As to ion regulation, MeHg at ≥10 ppb decreased the densities of skin stem cells and ionocytes, leading to declines in ion (Na+, K+, and Ca2+) contents and H+/NH4+ excretion levels. A gene expression analysis also revealed declines in messenger RNA levels of several ion-regulatory genes (ncc2b, trpv6v1a, trpv5/6, ncx1b, and rhcg1). This study demonstrated that the lateral line sensory and ion regulatory functions of fish are extremely sensitive to MeHg.

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.

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.