Medaka Larvae Research Articles

Comparisons of analytical chemistry and biological activities of extracts from North Pacific gyre plastics with UV-treated and untreated plastics using in vitro and in vivo models

Plastic debris is an emerging worldwide threat to marine biota. Marine species may face unique challenges in low-flow estuarine systems with a high abundance of “macro-sized” (>4.75 mm) plastic due to the leaching of constituents and adsorbed contaminants. To simulate this leaching process, plastic samples recovered from the North Pacific Gyre along with corresponding UV-irradiated virgin plastic and non-irradiated virgin plastic counterparts were incubated in saltwater for 30 days at ambient temperatures ranging from 17 to 25 °C. Following solid-phase extraction, water samples were fractionated with sequential methanol elution from 10 to 100% and evaluated using in vitro assays assessing estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) activities. In vivo responses (vitellogenin [vtg] and cytochrome p450 1A [cyp1a] mRNA) were measured following 5-day exposures in Japanese medaka (Oryzias latipes) larvae (3 days post hatch). Estrogenic plasticizers, co-planar PCBs and PAHs were quantified in the extracts using targeted GC–MS/MS and UPLC-MS/MS. In vitro estrogenicity showed highest activity in the 70% methanol fraction for all plastic leachate exposures. Whole extract in vitro estradiol equivalent (EEQ) values were 4.34 ± 2.65, 8.79 ± 2.09 and 13.78 ± 3.64 ng/L, for virgin plastic, UV-irradiated virgin plastic and North Pacific Gyre-recovered plastic, respectively (mean ± SD). Significant vtg induction was observed in medaka larvae exposed to leachate extracts from North Pacific Gyre-recovered plastic and UV-irradiated virgin plastic (9.9-fold, p = 0.039 and 10.1-fold, p = 0.042, respectively). Chemically-determined EEQ values were also localized in the 70% methanol fraction. Whole leachate extract chemical EEQ values were 0.33 ± 0.07, 1.64 ± 0.62 and 11.4 ± 2.13 ng/L, for virgin plastic, UV-irradiated virgin plastic and North Pacific Gyre-recovered plastic, respectively. In-vitro AhR activity was highest in the 70% methanol elution with greater activity in North Pacific Gyre-recovered plastic than in virgin plastic and UV-irradiated virgin plastic (toxic equivalency [TEQ] = 1.06 ± 0.54, 0.38 ± 0.07 and 0.71 ± 0.47 ng/L, respectively). CYP1A mRNA was significantly induced in larval medaka exposed to North Pacific Gyre-recovered plastic leachates (17.8-fold, p = 0.02) while exposure to virgin plastic and UV-irradiated virgin plastic leachates caused no significant change. Chemically-determined TEQ analysis for AhR indicated highest activity in the 90% methanol fraction for all leachates, with whole extract in vitro TEQs being 1.47 ± 0.87, 0.03 ± 0.05 and 0.42 ± 0.38 ng/L for North Pacific Gyre-recovered plastic, virgin plastic and UV-irradiated virgin plastic, respectively. These results indicate that weathering and UV radiation release estrogenic plasticizers and demonstrate the ability for plastics to transport adsorbed persistent organic pollutants at eco-toxicologically relevant concentrations.

Establishment of a quantitative in vivo method for estimating adipose tissue volumes and the effects of dietary soy sauce oil on adipogenesis in medaka, Oryzias latipes.

Adipose tissue, which is conserved in higher eukaryotes, plays central roles in controlling the body’s energy balance, including excess energy storage and energy expenditure during starvation. In adipogenesis, intranuclear receptor, peroxisome proliferator–activated receptor gamma (PPARγ) is a key molecule, and PPARγ agonists can promote adipogenesis. Many studies on the in vitro screening of PPARγ agonists with compounds derived from various materials have been reported; however, in vivo assays for quick examination of these feeding effects have not been established. In this study, we developed a technique using a lipophilic fluorescent reagent, Nile red to quantitatively estimate the adipose tissue volumes by using Japanese rice fish, medaka (Oryzias latipes) and studied effects of dietary soy sauce oil (SSO), which is a discarded by-product from Japanese traditional food and is known to have PPARγ-agonistic activity, on adipogenesis. We found that SSO feeding increased the adipose tissue volumes, and the expression levels of adipogenesis-related genes increased in these medaka larvae. These results suggest that SSO feeding increases the adipose tissue volumes through adipogenesis promotion by PPARγ-agonistic activity in medaka, and medaka is a powerful model for studying adipogenesis. Furthermore, our study also demonstrates the availability of SSO as a dietary additive for farmed fish.

Micro-elemental retention in rotifers and their trophic transfer to marine fish larvae: Influences of green algae enrichment

Application of “green water” has been widely practiced in larval fish aquaculture, whereas their impacts on micro-elemental nutrition of rotifers and marine fish larvae have been seldom investigated. In the present study, we evaluated the impacts of “green water” and “clean water” on the micro-elemental changes and profiles in rotifers (both S- and L- types), and their subsequent impacts on marine fish larvae. In “clean water”, the gut contents (unfinished algae) of rotifers were lost rapidly. However, the application of “green water” relieved the rapid loss of all microelements (especially for Mn and Co), with more profound effects observed on the S-type rotifers. Under long-term enriched conditions, Cu and Zn in rotifers were rapidly lost in “clean water”, and the application of “green water” even facilitated the loss of all three enriched micro-elements (i.e., Cu, Zn, and Se). We demonstrated that the inclusion of gut contents (algae) in rotifers provided more bioaccessible micro-elements (>80% in the non-skeleton fraction) than the empty-gutted rotifers. Furthermore, these gut contents (algae) in rotifers also provided more bioavailable Fe source by increasing its assimilation efficiency in both 5 and 25 dph medaka larvae. In conclusion, the present study showed the positive effects of “green water” on the micro-elemental nutrition in rotifers and larval fish. However, their negative impacts on the enriched micro-elements in rotifers should also be considered during real application.

Combined effects of ocean acidification and crude oil pollution on tissue damage and lipid metabolism in embryo-larval development of marine medaka (Oryzias melastigma).

Ocean acidification (OA) and crude oil pollution have been highlighted as some of the most pervasive anthropogenic influences on the ocean.In marine teleosts, early life-history stages are particularly vulnerable to disturbance by CO2-driven acidification as they lack pH-mediated intracellular regulation. Embryos exposed to trace levels of crude oil constituents dissolved in water exhibit a common syndrome of developmental abnormalities. So far, little is known about the combined effects of OA and crude oil on the early life history of marine fish. Eggs and larvae of the marine medaka (Oryzias melastigma) were treated with CO2 (1080μatm atmospheric CO2), the water-soluble fraction (WSF) of crude oil (500μg/L) and a CO2 (1080μatm atmospheric CO2)/WSF (500μg/L) mixture within 4h after oviposition. Isolated and combined OA/WSF had no detectable effect on embryonic duration, egg survival rate and size at hatching. Histopathological anomalies of tissue and lipid metabolic disorder were significant when CO2 or WSF was given alone at 30days of age. Combination of CO2 and WSF enhanced their toxicity compared to their separate administration. Since the early life-history stage of marine fish is thought to be impacted more heavily by increasing CO2 partial pressure (pCO2) levels and crude oil pollution, OA and crude oil pollution have the potential to act as an additional source of natural mortality.

Toxicity implications for early life stage Japanese medaka (Oryzias latipes) exposed to oxyfluorfen.

We investigated the potential toxic effects of Oxyfluorfen (OXY), an herbicide used in agriculture, on the embryo-larval development of Japanese medaka fish (Oryzias latipes). Embryos (1-day postfertilization) and larvae (2-day posthatch) were exposed to OXY (0.5-8 mg/L) for 96 h and evaluated for mortality and hatching on embryos, and the mortality and growth on larvae during depuration. It was observed that the embryo-mortality was inconsistently altered by OXY; only the 2 mg/L group showed significant reduction on embryo survivability. However, larval-mortality was concentration-dependent and OXY exposure induced scoliosis-like phenotypic features in the surviving larvae; the calculated LC50 was 5.238 mg/L. Our data further indicated that larval skeleton, both axial and appendicular, was the potential target site of OXY. Skeletal growth in larvae exposed to 2 mg/L was inhibited significantly until 1 week of depuration with regard to the linear lengths of neurocranium, Meckel's cartilage, caudal vertebrae (first 10) in the axial skeletons, and pectoral fin and urostyle in the appendicular skeletons. Moreover, the total protein content remained unaltered by OXY after 96 h exposure; while the RNA concentration was reduced significantly in larvae exposed to 2 mg/L. Expression analysis of several genes by quantitative real-time RT-PCR (RT-qPCR) showed significant upregulation of zic5, a zinc-finger type transcription regulator, at the transcription level. This study indicated that the scoliosis induced by OXY in Japanese medaka larvae was the result of stunted skeletal growth, probably because of deregulation of zinc-finger type transcription regulators, at the genomic level.

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.

Illicit drug ketamine induces adverse effects from behavioral alterations and oxidative stress to p53-regulated apoptosis in medaka fish under environmentally relevant exposures.

With increasing problems of drug abuse worldwide, aquatic ecosystems are contaminated by human pharmaceuticals from the discharge of hospital or municipal effluent. However, ecotoxicity data and related toxic mechanism for neuroactive controlled or illicit drugs are still lacking, so assessing the associated hazardous risk is difficult. This study aims to investigate the behavioral changes, oxidative stress, gene expression and neurotoxic or apoptosis effect(s) in larvae of medaka fish (Oryzias latipes) with environmentally relevant exposures of ketamine (KET) solutions for 1-14 days. KET exposure at an environmentally relevant concentration (0.004μM) to 40μM conferred specific patterns in larval swimming behavior during 24h. At 14 days, such exposure induced dose- and/or time-dependent alteration on reactive oxygen species induction, the activity of antioxidants catalase and superoxide dismutase, glutathione S-transferase and malondialdehyde contents in fish bodies. KET-induced oxidative stress disrupted the expression of acetylcholinesterase and p53-regulated apoptosis pathways and increased caspase expression in medaka larvae. The toxic responses of medaka larvae, in terms of chemical effects, were qualitatively analogous to those of zebrafish and mammals. Our results implicate a toxicological impact of waterborne KET on fish development and human health, for potential ecological risks of directly releasing neuroactive drugs-containing wastewater into the aquatic environment.

Differential sensitivities to dioxin-like compounds PCB 126 and PeCDF between Tg(cyp1a:gfp) transgenic medaka and zebrafish larvae

It has been intensively documented that there are species-differences in the sensitivity to dioxin-like compounds (DLCs) in mammalian and avian. However, this issue is still unclear in fish. This study aimed at evaluating the differential sensitivities to DLCs in fish larvae. Here, larvae of Tg(cyp1a:gfp) medaka and Tg(cyp1a:gfp) zebrafish were tested with 2,3,7,8-Tetrachlorodibenzodioxin (TCDD), polychlorinated biphenyl 126 (PCB 126) and 2,3,4,7,8,-Pentachlorodibenzofuran (PeCDF). Comparative analyses were performed on induction of GFP fluorescence, expression of endogenous cyp1a mRNAs and EROD activity between the two species after exposure to these chemicals. We found that PCB 126 and PeCDF exposure at high concentrations induced strong GFP expression in multiple organs (liver, head kidney and gut) in both medaka and zebrafish larvae. Moreover, the expression of endogenous cyp1a mRNA was significantly elevated in the zebrafish larvae exposed to TCDD, PCB 126 and PeCDF at different concentrations. Likewise, almost all the exposure conditions could cause prominent elevation of EROD activity in the zebrafish larvae, while the EROD activities were just slightly elevated in the medaka larvae exposed to 1 nM and 0.5 nM of TCDD as well as to 1.5 nM and 15 nM of PeCDF, but not in the medaka larvae exposed to PCB 126. Taken together, zebrafish was proved to be more sensitive than medaka to PCB 126 and to PeCDF in this study. The findings suggested species-specific sensitivity to DLCs in fish and will facilitate choosing a sensitive and reliable fish model or tool to evaluate the risk of dioxins and DLCs exposure.

Developmental ethanol exposure impairs locomotor movement in Japanese medaka (Oryzias latipes) larvae targeting epigenome

Evidence indicated ethanol exposure during development disrupts brain functions that induces fetal alcohol spectrum disorder (FASD) phenotypes with behavioral abnormalities. We aimed to investigate whether prenatal ethanol exposure has any potential impact on behavior of a FASD fish model. Fertilized Japanese medaka (Oryzias latipes) eggs were exposed to 100–300 mM ethanol or 2 mM 5-azacytidine (5-azaC), 0–2 day post fertilization (dpf), in embryo-rearing medium (ERM). Survived embryos were maintained in clean ERM and used either for gene expression analysis on 2- and 6-dpf or allowed to hatch for behavioral study. Photomotor response of 3–4 day post hatch larvae were tracked for 3 h with light-dark transitions. It was observed that larval swimming was phototactic; enhanced in presence of light, declined in dark. Phototactic response was also observed in larvae prenatally exposed to ethanol or 5-azaC; however, the total distance swum by these larvae compared to controls declined. Further analysis indicated that, in light phases, total swimming activity and average swimming speed were reduced in larvae prenatally exposed to ethanol (300 mM) or 5-azaC. Expression analysis of baz1a and baz2a in embryos indicated developmental regulation. Ethanol (100–300 mM) or 5-azaC (2 mM) were able to modulate downregulation of both baz1a and baz2a mRNAs only in 6 dpf embryos of 300 mM ethanol and 5-azaC (2 mM) groups. These studies indicated that prenatal exposure to ethanol or 5-azaC was able to disrupt movements and thus swimming behavior in FASD phenotypes probably due to delayed remodeling of genome and epigenome.

Functional analyses of a novel vesicular membrane protein, TMEM141 during the growth stage in medaka (Oryzias latipes) larvae

Functional analyses of a novel vesicular membrane protein, TMEM141 during the growth stage in medaka (Oryzias latipes) larvae

Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae

We investigated the effect of combined exposure to nodavirus infection and TBT on medaka (Oryzias latipes). Medaka larvae were infected by immersion in medium containing nodavirus at titers of 102.5, 103.5, or 104.5 TCID50/mL. Infected fish then were exposed to TBT at 0, 0.17, 0.52, 1.6, or 4.7μg/L. Of the 12 groups exposed to both stressors, the mortalities of 6 (102.5 TCID50/mL+0.52, 1.6, or 4.7μg/L, 103.5 TCID50/mL+4.7μg/L and 104.5 TCID50/mL+1.6 or 4.7μg/L) were significantly higher than that of each TBT control. Specifically, mortality was 46±5.5% in the group exposed to both 102.5 TCID50/mL virus and 0.52μg/L TBT, which represent the lowest observed effective dose and concentration, respectively, among the 6 groups with increased mortalities. Our results suggest that combined exposure to both stressors suppresses antiviral mechanisms in the fish, thus increasing mortality.

Developmental toxicity and molecular responses of marine medaka (Oryzias melastigma) embryos to ciguatoxin P-CTX-1 exposure

Ciguatoxins are produced by toxic benthic dinoflagellates and cause ciguatera fish poisoning worldwide, but the toxic effects on developing marine fish have not been well investigated. The Pacific ciguatoxin (P-CTX-1), is a potent sodium channel agonist, which is one of the most toxic members among all CTXs. This study evaluated the toxic effects of microinjecting purified Pacific ciguatoxin-1 (P-CTX-1) on embryonic development of marine medaka Oryzias melastigma. A lower 96h-LD50 value was estimated for eleuthero-embryos (1.32ngg−1) than that for embryos (1.71ngg−1), indicating that P-CTX-1 is more lethal to newly hatched medaka larvae. P-CTX-1 induced detrimental effects during embryonic development, including hatching failure, abnormalities in physical development (caudal fin malformation and spinal deformities), internal damage (green coloration of the gall bladder and hemorrhaging), immune dysfunction, and altered muscle physiology (bradycardia and hyperkinetic twitching). The results of a transcriptional expression analysis of genes related to the stress/immune responses, cardiac and bone development, and apoptosis supported the observed developmental abnormalities. This study advanced the understanding of P-CTX-1 mediated toxic mechanisms in the development of early life stages of a fish, and thus contributed to the toxicity assessment of CTXs in marine ecosystems.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor κB Ligand (RANKL) under the control of a heatshock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Ancestral benzo[a]pyrene exposure affects bone integrity in F3 adult fish (Oryzias latipes)

Benzo[a]pyrene (BaP) at an environmentally relevant concentration (1μg/L) has previously been shown to affect bone development in a transgenerational manner in F3 medaka (Oryzias latipes) larvae (17dph). Here, we provide novel histomorphometric data demonstrating that the impaired bone formation at an early life stage is not recoverable and can result in a persistent transgenerational impairment of bone metabolism in F3 adult fish. A decrease in bone thickness and the occurrence of microcracks in ancestrally BaP-treated adult male fish (F3) were revealed by MicroCt measurement and histopathological analysis. The expression of twenty conserved bone miRNAs were screened in medaka and their relative expression (in the F3 ancestral BaP treatment vs the F3 control fish) were determined by quantitative real-time PCR. Attempt was made to link bone miRNA expression with the potential target bone mRNA expression in medaka. Five functional pairs of mRNA/miRNA were identified (Osx/miR-214, Col2a1b/miR-29b, Runx2/miR-204, Sox9b/miR-199a-3p, APC/miR-27b). Unique knowledge of bone-related miRNA expression in medaka in response to ancestral BaP-exposure in the F3 generation is presented. From the ecological risk assessment perspective, BaP needs to be regarded as a transgenerational skeletal toxicant which exerts a far-reaching impact on fish survival and fitness. Given that the underlying mechanisms of cartilage/bone formation are conserved between medaka and mammals, the results may also shed light on the potential transgenerational effect of BaP on skeletal disorders in mammals/humans.

Toxicokinetics and toxicodynamics of chlorpyrifos is altered in embryos of Japanese medaka exposed to oil sands process-affected water: evidence for inhibition of P-glycoprotein.

Oil sands process-affected water (OSPW) is generated during extraction of bitumen in the surface mining oil sands industry in Alberta, Canada. Studies were performed in vitro by use of Caco-2 cells, and in vivo with larvae of Japanese medaka (Oryzias latipes) to determine if organic compounds from the aqueous phase of OSPW inhibit ATP binding cassette protein ABCB1 (permeability-glycoprotein, P-gp). Neutral and basic fractions of OSPW inhibited activity of P-gp in Caco-2 cells by 1.9- and 2.0-fold, respectively, while the acidic fraction had the least effect. The organophosphate pesticides chlorpyrifos (a substrate of P-gp) and malathion (not a substrate of P-gp), were used as model chemicals to investigate inhibition of P-gp in larvae. Co-exposure to chlorpyrifos and an extract of OSPW containing basic and neutral compounds reduced survival of larvae to 26.5% compared to survival of larvae exposed only to chlorpyrifos, which was 93.7%. However, co-exposure to malathion and the extract of OSPW did not cause acute lethality compared to exposure only to malathion. Accumulation and bioconcentration of chlorpyrifos, but not malathion, was greater in larvae co-exposed with the extract of OSPW. The terminal elimination half-life of chlorpyrifos in larvae exposed to chlorpyrifos in freshwater was 5 days compared with 11.3 days in larvae exposed to chlorpyrifos in OSPW. Results suggest that in non-acute exposures, basic and neutral organic compounds in the water-soluble fraction of OSPW inhibit activity of P-gp, which suggests that OSPW has the potential to cause adverse effects by chemosensitization. Copyright © 2016 John Wiley & Sons, Ltd.

Dissolved Organic Matter or Salts Change the Bioavailability Processes and Toxicity of the Nanoscale Tetravalent Lead Corrosion Product PbO2 to Medaka Fish.

Nanoscale lead dioxide (nPbO2(s)) is a corrosion product formed from the chlorination of lead-containing plumbing materials. This metal oxide nanoparticle (NP) plays a key role in determining lead pollution in drinking water and receiving water bodies. This study uses nPbO2(s) and medaka fish (Oryzias latipes) as surrogates to investigate the aqueous fate and toxicological risk of metal oxide NPs associated with water matrices. The larvae of medaka were treated with solutions containing nPbO2(s) or Pb(II)aq in different water matrices for 7-14 days to investigate the in vivo toxic effects of NPs. Ionic strength enhanced aggregation and sedimentation of nPbO2(s) in water, leading to increased lead contents in fish bodies. However, the presence of dissolved organic matter in water enhanced particle stability and accelerated the lead dissolution, thus changing the bioavailability processes (bioaccessibility) of particles. Oxidative stress response and neurotoxicity in exposed fish was greater for nPbO2(s) solution with increased salinity than dissolved organic matter. We predict the bioavailability processes and toxicity of nPbO2(s) in medaka from the aqueous particle behavior under environmentally relevant exposure conditions. Our investigation suggests a toxicological risk of metal oxide NP pollution in the aquatic environment.

Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting 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.

Developmental neurotoxicity of organophosphate flame retardants in early life stages of Japanese medaka (Oryzias latipes).

Because brominated flame retardants are being banned or phased out worldwide, organophosphate flame retardants have been used as alternatives on a large scale and have thus become ubiquitous environmental contaminants; this raises great concerns about their environmental health risk and toxicity. Considering that previous research has identified the nervous system as a sensitive target, Japanese medaka were used as an aquatic organism model to evaluate the developmental neurotoxicity of 4 organophosphate flame retardants: triphenyl phosphate, tri-n-butyl phosphate, tris(2-butoxyethyl) phosphate, and tris(2-chloroethyl) phosphate (TCEP). The embryo toxicity test showed that organophosphate flame retardant exposure could decrease hatchability, delay time to hatching, increase the occurrence of malformations, reduce body length, and slow heart rate. Regarding locomotor behavior, exposure to the tested organophosphate flame retardants (except TCEP) for 96 h resulted in hypoactivity for medaka larvae in both the free-swimming and the dark-to-light photoperiod stimulation test. Changes of acetylcholinesterase activity and transcriptional responses of genes related to the nervous system likely provide a reasonable explanation for the neurobehavioral disruption. Overall, the present study clearly demonstrates the developmental neurotoxicity of various organophosphate flame retardants with very different potency and contribute to the determination of which organophosphate flame retardants are appropriate substitutes, as well as the consideration of whether regulations are reasonable and required. Environ Toxicol Chem 2016;35:2931-2940. © 2016 SETAC.

Three-dimensional visualization of green fluorescence protein-labelled Edwardsiellatarda in whole Medaka larvae.

The invasive fish pathogen Edwardsiellatarda is common in aquatic environments and causes the environmentally and economically destructive emphysematous putrefactive disease called edwardsiellosis. In order to understand the organism's infection pathway, medaka larvae (Oryziaslatipes) were immersion-infected with E.tarda labelled with green fluorescence protein (GFP) and then visualized in three dimensions under confocal laser microscopy and light-sheet fluorescence microscopy. Confocal microscopy revealed GFP-labelled E.tarda in the mouth, head, gill bridges, gill cover, skin, membrane fin, gastrointestinal tract and air bladder, and in the caudal vein, somite veins, caudal artery and caudal capillaries. Light-sheet microscopy additionally showed GFP-labelled E.tarda in the pharyngeal cavity, muscle of the pectoral fin and cardiac atrium and ventricle. These findings suggest that during its infection of fish, E.tarda initially adheres to, and invades, the epithelial cells of the skin, gills and gastrointestinal tract (through the pharyngeal cavity); E.tarda then enters the blood vessels to access organs, including the air bladder and heart.

Silver nanoparticle toxicity is related to coating materials and disruption of sodium concentration regulation

Silver nanoparticles (AgNPs) have been increasingly commercialized and their release into the environment is imminent. Toxicity of AgNP has been studied with a wide spectrum of organisms, yet the mechanism of toxicity remains largely unknown. This study systematically compared toxicity of 10 AgNPs of different particle diameters and coatings to Japanese medaka (Oryzias latipes) larvae to understand how characteristics of AgNP relate to toxicity. Dissolution of AgNPs was largely dependent on particle size, but their aggregation behavior and toxicity were more dependent on coating materials. 96 h lethal concentration 50% (LC50) values correlated with AgNP aggregate size rather than size of individual nanoparticles. Of the AgNPs studied, the dissolved Ag concentration in the test suspensions did not account for all of the observed toxicity, indicating the role of NP-specific characteristics in resultant toxicity. Exposure to AgNP led to decrease of sodium concentration in the tissue and increased expression of Na+/K+ ATPase. Gene expression patterns also suggested that toxicity was related to disruption of sodium regulation and not to oxidative stress.