Oryzias Melastigma Research Articles

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method

The choriogenin H – EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17β-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

Triphenyltin exposure causes changes in health-associated gut microbiome and metabolites in marine medaka

Triphenyltin (TPT), an organic compound with a wide range of applications, is often detected in water bodies and aquatic animals. However, the mechanism underlying the biological metabolic health problems caused by long-term exposure to environment concentrations of TPT remains unclear. The morphology and gene expression in the gut and liver were investigated; and 16SrRNA gene amplification sequencing and non-targeted LC-MS/MS metabonomics were investigated after marine medaka (Oryzias melastigma) was treated with 1, 10, and 100 ng/L TPT for 21 days. During prolonged exposure to TPT, the adaptation mechanism maximized the energy of absorption, increased the length of intestinal microvilli, reduced the number of rough endoplasmic reticulum in the liver, and caused loss of weight. TPT exposure significantly changed the intestinal microbiome of marine medaka, thereby resulting in a significant decrease in microbial diversity. Following exposure to 100 ng/L TPT, the metabolic profiles were significantly changed and the altered metabolites were mainly concentrated in the lipid metabolic pathway. Finally, based on comprehensive network analysis, the association between the significantly changed bacteria and metabolites contributed further to the prediction of the impact of TPT on the host. This study provides a novel insight into the underlying mechanisms of host metabolic diseases caused by TPT and emphasizes the importance of monitoring pollutants in the environment.

Ultrasensitive label-free electrochemical immunosensors for detecting marine medaka (Oryzias melastigma) vitellogenin based on novel Cu2O–BSA nanoparticles and anti-lipovitellin monoclonal antibody

Vitellogenin (Vtg) is a biomarker for environmental estrogens, and its sensitive detection method is vital for the detection of weak estrogenic activity. This study aimed to develop sensitive electrochemical immunosensors for marine medaka (Oryzias melastigma) Vtg based on novel nanomaterials. Firstly, Cu2O–BSA nanoparticles (NPs) with a diameter of 120−150 nm was synthesized, and monoclonal antibody against lipovitellin (anti-Lv mAb) was prepared. Then two simple and efficient label-free immunosensors for the detection of Vtg were established using Cu2O–BSA NPs and gold nanoparticle (Au NPs) as signal amplification elements and anti-Lv mAb as the detection probe. These two immunosensors have good sensitivity, selectivity, reproducibility for Vtg quantification, and are easy to use. Moreover, the BSA/Anti-Lv mAb/Au/Cu2O–BSA/GCE immunosensor exhibited a linear range from 0.128 to 5 × 104 pg/mL, with the lowest detection limit (LOD) of 0.09 pg/mL, which is considerably lower than those of the previously reported Vtg detection techniques. Finally, the performance of the immunosensor was evaluated by quantifying Vtg concentrations in plasma of male marine medaka exposed three estrogenic bisphenols. The results demonstrated that the BSA/Anti-Lv mAb/Au/Cu2O–BSA/GCE immunosensor was an easily-operated, ultrasensitive and accurate detection method for weak estrogenic activity.

Generation of a novel transgenic marine medaka (Oryzias melastigma) for highly sensitive detection of heavy metals in the environment

As typic priority pollutants in the marine environment, heavy metals can be accumulated in the human body leading to serious environmental and health problems. The metal regulatory elements (MREs) have been identified to be the main functional parts for the response to heavy metals. To develop a convenient biological monitoring tool for the detection of heavy metals in the oceans, we generated a transgenic marine medaka line Tg(OmMT: eGFP) with a truncated metallothionein promoter, which was only 193 bp and drove the expression of eGFP. After Tg(OmMT:eGFP) embryos were treated with four different heavy metals and different concentrations, the results showed that the expression level of eGFP was consistent with that of the endogenous mt. The transgenic embryos are very sensitive to Hg2+, and the fluorescence could be induced in the 0.0002 μM concentration, which is far lower than the primary water standard. The expression level of eGFP and mt showed a dose-dependent manner to heavy metals concentration. Taken together, the newly established marine medaka is a sensitive, efficient, and convenient tool for monitoring heavy metal pollution in the environment, especially seawater.

Influence of salinity on physiological development and zinc toxicity in the marine medaka Oryzias melastigma.

To determine whether the marine medaka Oryzias melastigma is a suitable model organism for evaluating the effects of environmental chemicals on marine teleosts, we examined the effect of salinity on physiological development and zinc toxicity. Growth as measured by total body length was significantly lower in fresh water compared to brackish water. Reproductive success was also significantly reduced in fresh water, although we observed cells in the pituitary producing gonadotropins such as Gpa (common glycoprotein hormone α), Fshb (follicle stimulating hormone β), and Lhb (luteinizing hormone β) at all salinities. These results indicate that O. melastigma is adaptable to various salinities from fresh to seawater, and brackish water is best for physiological processes including growth performance and reproduction. When zinc was dissolved in saltwater, a white precipitate formed immediately, and the dissolved concentration decreased in the supernatant and increased at precipitate. We performed zinc toxicity tests on early life stage and adult stage in fresh water, brackish water, and seawater. Among adults, the lowest observed effect concentration for mortality in freshwater (15.3 mg/L) was lower than in brackish water (>48 mg/L) or seawater (>48 mg/L). Similarly, among embryos and larvae, the lowest observed effect concentration for mortality in freshwater (4.8 mg/L) was lower than in brackish water (48 mg/L) or seawater (48 mg/L). These results highlight the importance of using marine organisms to evaluate the ecological effects of marine pollutants.

Proteomic Response of the Brain to Hypoxic Stress in Marine Medaka Fish (Oryzias melastigma)

Hypoxia is a serious issue that affects the marine environment, with a growing number of hypoxic “dead zones” occurring each year. Reports have indicated that hypoxia is detrimental to the reproductive function and sexual development of fish via the disruption of endocrine signaling in organs involved in the hypothalamus–pituitary–gonad axis, including the brain. While we previously reported that hypoxia induces transcriptome-wide alterations in the brain of marine medaka (Oryzias melastigma), whether these effects were reflected at the protein level remains unclear. Therefore, the present study used high-throughput proteomic sequencing along with bioinformatics analysis to assess the short-term and multi-generational effects of hypoxia on the brain proteome of O. melastigma. We identified 36,567 peptides and 7,599 proteins (1% false discovery rate in brain samples), with functions involved in cellular and metabolic processes such as signaling and reproductive processes as well as energy production and conversion. Furthermore, we determined that hypoxia resulted in the significant differential expressions of 33 upregulated and 69 downregulated proteins in the short-term exposure group and 24 upregulated and 52 downregulated proteins in the multi-generational exposure group. Pathway enrichment analysis of the deregulated proteins indicated that hypoxia could impair brain function by altering arachidonic acid metabolism, tight junctions, and adrenergic signaling under short-term hypoxic exposure and by altering p53 and PI3K–Akt signaling under multi-generational hypoxic exposure, which may lead to the onset of neurodegenerative disorders including Alzheimer’s disease and amyotrophic lateral sclerosis. Ingenuity pathway analysis of the deregulated proteins showed that hypoxia affected common signaling pathways in the brain (e.g., integrin, paxillin, and epithelial adherens junction signaling) under both short-term and multi-generational exposures. Hypoxia also deregulated pathways specific to short-term exposure (including integrin-linked kinase, calcium, and integrin signaling) and multi-generational exposure (including sphingosine-1-phosphate signaling, endocannabinoid neuronal synapse pathway, and endoplasmic reticulum stress pathway). Overall, our results provide additional insights into the mechanisms of hypoxia disrupting neuronal function at the protein level in marine medaka.

Developmental toxicity in marine medaka (Oryzias melastigma) embryos and larvae exposed to nickel

As an important trace metal, nickel (Ni) has been reported extensively in the studies on freshwater animals. However, the toxic effects of Ni on marine organisms are not clearly understood. Therefore, in order to investigate the toxic effects of Ni on the early development of marine fish, the marine medaka (Oryzias melastigma) embryos and larvae were immersed in 0.13–65.80 mg/L Ni solution. The results showed that Ni exposure changed the egg size and heart rate of the embryos, lowered the hatchability, increased the deformity rate, and shortened the total body length of newly hatched larvae. Besides, it was found that before organogenesis and post-hatching periods were the sensitive periods of embryos to Ni. The 25 d LC50 value of embryos was 49.28 mg/L, and the 5 d LC50 of larvae was 55.92 mg/L, indicating that the embryos were more sensitive to Ni than the larvae. Furthermore, the expressions of the metallothionein (MT) gene, the skeletal development-related gene (Cyp26b1) and the cardiac development-related genes (ATPase, smyd1, cox2 and bmp4) were determined, and the results showed that the expressions of ATPase and smyd1 were up-regulated, while MT, Cyp26b1 and cox2 were significantly down-regulated at 9 days post-fertilization (dpf). Overall, Ni exposure caused a significant toxic effect on the early development of the O. melastigma embryos and larvae. Our findings could provide an important supplement to the toxicity data of tropical Ni and provide a reference for the exploration of the molecular mechanisms of Ni toxicity.

Identification and functional analysis of the perforin-1 like gene in disease resistance in half smooth tongue sole (Cynoglossus semilaevis)

The pore-forming protein perforin is one of the effectors of cell-mediated killing via the granule exocytosis pathway. In this study, a genome-wide association study was conducted in Vibrio harveyi disease-resistant and disease-susceptible families of half smooth tongue sole (Cynoglossus semilaevis) to determine the genes accounting for host resistance, and a perforin homologue was identified, designated perforin-1 like (CsPRF1l). The full-length cDNA of CsPRF1l is 1835 bp, and encodes 514 amino acids. The CsPRF1l gene consists of 10 exons and 9 introns, spanning approximately 7 kb. The amino acid sequence of CsPRF1l shows 60.35, 54.03, 41.92, and 34.17% identities to Morone saxatilis PRF1l, Oryzias melastigma PRF1l, Danio rerio PRF1.5 and Homo sapiens PRF, respectively. Sequence analysis revealed the presence of membrane attack complex/perforin (MACPF) and C2 domains in CsPRF1l. Quantitative real-time PCR showed that CsPRF1l presented a higher intestinal expression level in disease-resistant families than in susceptible families. Tissue expression pattern analysis showed that CsPRF1l is present in most of the tested tissues and highly expressed in the intestine, brain, stomach and gills. After challenge with V. harveyi, CsPRF1l mRNA was markedly upregulated in the liver, spleen, kidney, intestine, gills and skin. In addition, the recombinant CsPRF1l protein exhibited obvious antimicrobial activity against V. harveyi in vitro and in a zebrafish model. Collectively, these data indicate that CsPRF1l modulates host immune defense against V. harveyi invasion and provide clues about the efficacy of rCsPRF1l in fish that will give rise to useful therapeutic applications for V. harveyi infection in C. semilaevis.

First observation of conjoined twins in Oryzias melastigma.

First observation of conjoined twins in Oryzias melastigma.

Adaptation of life-history traits and trade-offs in marine medaka (Oryzias melastigma) after whole life-cycle exposure to polystyrene microplastics

Microplastics are ubiquitous in marine environments and may cause unexpected ecological effects. This study adopted a whole life-cycle exposure to illuminate the impact of polystyrene microplastics on life-history strategies of marine medaka (Oryzias melastigma), including the hatching of embryos, growth and reproduction of F0 generation, and embryonic and larval development of F1 offspring. Microplastics accumulated on the eggshell and reduced embryonic hatching rate and larval body length and weight. Similarly, 150 days of microplastic exposure decreased body mass and gonadosomatic index of adult fish, but accelerated sexual maturity of female fish, showing a trade-off between growth and reproduction. Microplastic exposure also caused obvious histopathological damages to gonads and decreased egg productions and fertilization rates. Moreover, parental microplastic exposure induced elevated heartbeats, premature hatching, and slow growth in F1 offspring. Anti-oxidative stress response, sex hormone disruption, and disturbed transcription of steroidogenic genes in the reproductive axis could partially explain the reproduction impairment and transgenerational trade-offs. Furthermore, transcriptome analysis revealed that the steroid hormone biosynthesis and cytochrome P450 pathways in the testes of male fish were significantly affected after 20 μg/L microplastic exposure. These findings suggest that microplastic pollution may be an emerging threat to the sustainability of marine fish population.

Size-dependent adverse effects of microplastics on intestinal microbiota and metabolic homeostasis in the marine medaka (Oryzias melastigma)

Microplastic (MP) is an emerging environmental pollutant and exposure to MPs has been associated with numerous adverse health outcomes in both wild and laboratory animals. The toxicity of MPs depends on concentration, exposure time, chemical composition and size distribution, but the impacts of particle size remain inconclusive yet. In this study, adult marine medaka (Oryzias melastigma) were exposed to different size of polystyrene MPs (PS-MPs) with concentration of 10 mg/L for 60 days and the growth performance, lipid metabolism, immune parameters and gut microbiome were determined. Results indicated that particle size is a dominant factor causing lipid metabolism disorders and hepatic toxicity in PS-MPs-exposed fish. The bodyweight, adipocyte size and hepatic lipid contents were significantly increased in 200 μm PS-MPs-exposed fish, while 2 and 10 μm PS-MPs-exposed fish exhibited liver injury principally manifested asthepresence oflittlefibrosis and inflammation. Given that larger particles could not enter the circulatory system, the impacts of PS-MPs on intestinal microbial biota homeostasis were further investigated. The results not only showed the characterization of gut microbial communities in Oryzias melastigma, but also indicated that microbial diversity and composition were altered in gut of fish exposed to PS-MPs, in particular 200 μm PS-MPs. The differentially abundant bacterial taxa in PS-MPs-exposed fish mainly belonged to the phylum Verrucomicrobia, Firmicutes and Fusobacteria. And furthermore, increased abundance of Verrucomicrobia and Firmicutes/Bacteroidetes ratio and decreased Fusobacteria were correlated with the increased bodyweight. Intestinal microbiome should play a critical role in regulating host lipid metabolism in fish exposed to lager size of PS-MPs.

Toxicity effects of hydrophilic algal lysates from Coolia tropicalis on marine medaka larvae (Oryzias melastigma)

Coolia tropicalis is a species of benthic and epiphytic toxic algae, which can produce phycotoxins that intoxicate marine fauna. In this study, the potential toxic effects of C. tropicalis on fish were investigated using larval marine medaka (Oryzias melastigma) as a model to evaluate fish behavior, physiological performance, and stress-induced molecular responses to exposure to two sublethal concentrations (LC10 and LC20) of hydrophilic algal lysates. Exposure to C. tropicalis lysates inhibited swimming activity, activated spontaneous undirected locomotion, altered nerve length ration, and induced early development abnormalities, such as shorter eye diameter, body as well as axon length. Consistent with these abnormalities, changes in the expression of genes associated with apoptosis (CASPASE-3 and BCL-2), the inflammatory response (IL-1β and COX-2), oxidative stress (SOD), and energy metabolism (ACHE and VHA), were also observed. This study advances our understanding of the mechanisms of C. tropicalis toxicity in marine fish in the early life stages and contributes to future ecological risk assessments of toxic benthic dinoflagellates.

The adverse effects of acrylamide exposure on the early development of marine medaka (Oryzias melastigma) and its mechanisms

Acrylamide (AA) can have deleterious effects on freshwater fish. However, its adverse effects on euryhaline fish are still unknown. In this study, embryos of Oryzias melastigma were exposed to different concentrations of AA to investigate its effect on early developmental disorders. After 21 days of exposure, AA significantly inhibited the hatching rate and delayed the hatching time of embryos, and led to developmental delay, teratogenesis, and locomotion impairments in larvae. RNA-sequencing data of larvae indicated that AA upregulated the expression of hemoglobin and myoglobin involved in oxygen transport and angiopoietin 1, integrin, and matrix metallopeptidases related to angiogenesis and downregulated the expression of early growth response genes and synaptotagmin-2 related to neural plasticity and neurotransmitter release. Overall, our study showed that AA caused deleterious effects on the early development of euryhaline fish through hypoxic stress and neurotoxicity, providing a scientific basis for the environmental risk assessment of marine AA.

Probabilistic environmental risk assessment of microplastics in marine habitats

Microplastics are ubiquitous in the environment and given the large number of published hazard and exposure studies, quantitative environmental risks assessments of microplastics become feasible. We present here the first environmental risk assessment for marine waters based only on measured concentrations. The Thevariability and uncertainty of the measured data was accounted for in the exposure assessment, while probabilistic species sensitivity distributions were used for hazard assessment, from which a probability distribution was extracted for the predicted no-effect concentration (PNEC). By dividing the exposure distribution by the PNEC-distribution, we were able to calculate probabilistic risk characterisation ratios for each water body in which measurements were performed. Results show a good coverage of the world's major water bodies by measured exposure concentrations (MECs), while the hazard assessment could be improved by aligning the type of particles tested in hazard studies (size, form, polymer) to those actually found in the oceans. Overall, the mean predicted no-effect concentration (PNEC) is 3.84·106 part m−3, with Oryzias melastigma being the most sensitive species (calculated mean NOEC of 3.90·106 part m−3). Interestingly, the only type of dose descriptor that could be extracted from the literature for particles above 10−20 μm was the highest observed no effect concentration (HONEC), which indicates a very low or null toxicity of these larger MPs towards marine organisms. The mean MEC is 1.5·103 part m−3, the highest concentrations being measured in the Atlantic and Pacific Ocean. Although there is a very small overlap of the probability distribution associated with the RCR (0.00002 % of the data points), the mean RCR is 4·10-4 and therefore risks are unlikely given the available data. However, as increasing amounts of plastic reach the environment, RCRs can be expected to increase in the future.

Acute exposure of di(2-ethylhexyl) phthalate (DEHP) induces immune signal regulation and ferroptosis in oryzias melastigma

The plasticizer di (2-ethylhexyl) phthalate (DEHP) is becoming increasingly abundant throughout the global environment as plastic pollution becomes highly severe, especially in the ocean. The adverse effects of DEHP have garnered increasing concern as they are recognized as endocrine disruptors. However, information on the effects of DEHP in marine organisms remains limited. In this study, acute toxic effects on marine medaka (Oryzias melastigma) following DEHP exposure were investigated. Transcriptome analysis was performed on the livers of medaka exposed to DEHP for 6 and 24 h. Results showed that 1595 genes were affected in all the analyzed specimens, and several genes expressed variably according to sex. Some pathways associated with immunity, metabolism, and endocrine system were significantly enriched, with the complement system appearing to be the most affected immune pathway. Pathway enrichment indicated that, under acute DEHP exposure, the immune response of females tended to be more sensitive than that of males. In addition, ferroptosis occurred in response to DEHP exposure, which resulted in an enrichment of the ferroptosis pathway along with iron overload, an increase in malondialdehyde (MDA) and lipid peroxidation (LPO) content, and a decrease in glutathione (GSH) levels. These results indicate that a form of cell death characterized by iron-dependence occurred following DEHP exposure, but the underlying mechanism requires further analysis. This study implies that DEHP can alter some molecular regulation patterns within a short period and induce cell death through ferroptosis.

Biometric indices of five miniature fish from the catchment area of Kaptai Lake, Bangladesh

The present study reports on biometric indices comprising length-weight relationships (LWRs) of five miniature freshwater fish species from the Kaptai Lake, the largest lake in the People's Republic of Bangladesh. The study area included 3 stations in the 11,122 km2 catchment area of the lake and each station was sampled from November 2017 to April 2018 using push net (mesh size 2 mm) for capturing the species. The values of growth coefficient b of the LWRs (W = aTLb) of the species Oryzias dancena, Oryzias melastigma, Gobiopterus chuno, Brachygobius nunus and Badis chittagongis were 2.9575, 3.1467, 3.0535, 3.058 and 2.9396 with above 0.95 coefficient of correlation (r2). These results represent the first study of LWRs for these fish species which may help in management and conservation of the species in the whole country, including its geographical distribution.

Contact micropredation may play a more important role than exotoxicity does in the lethal effects of Karlodinium australe blooms: Evidence from laboratory bioassays

Multiple dinoflagellate species from the genus Karlodinium have been well known to form massive and toxic blooms that consequently cause fish kills in many coastal waters around the world. Karlodinium australe is a mixotrophic and potentially ichthyotoxic species associated with fish kills. Here, we investigated phagotrophy of K. australe (isolate KaJb05) established from a bloom event in the West Johor Strait, Malaysia, using several prey species (phytoplankton, zooplankton, and larval fish). The results showed that K. australe ingested relatively small prey cells of co-occurring microalgae by direct engulfment, while it fed on larger prey cells of microalgae by tube feeding. The results of animal exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval fish (Oryzias melastigma) demonstrated that phagotrophy (in terms of the trophic mode of the dinoflagellate), or micropredation (in terms of the mechanism of lethal effects on prey), played a more important role than the toxicity did in causing the lethal effects of K. australe on these aquatic animals under low cell densities of K. australe, while the mortalities of animals observed in the exposure to cell lysates of K. australe were solely caused by the toxicity. A comparison of the lethal effects between K. australe and K. veneficum revealed that the lethal effect of K. australe on rotifers was much stronger than that of K. veneficum at all cell densities applied in the experiments and the more "aggressive" micropredation of K. australe is suggested to explain the difference in lethal effect between K. austale and K. veneficum. Our results may explain why K. australe exhibited fish killings during moderate blooms at cell densities < 2.34 × 106 cells L−1, whereas K. veneficum was observed to cause massive fish kills only if the cell density was above 107 cells L−1. We believe these findings provide new insights into the ecological consequences of phagotrophy exhibited in some mixotrophic and harmful algae such as species of Karlodinium and of HAB events in general.

Germline Specific Expression of a vasa Homologue Gene in the Viviparous Fish Black Rockfish (Sebastes schlegelii) and Functional Analysis of the vasa 3' Untranslated Region.

Germ cells play a key role in gonad development. As precursors, primordial germ cells (PGCs) are particularly important for germline formation. However, the origination and migration patterns of PGCs are poorly studied in marine fish, especially for viviparous economic species. The vasa gene has been widely used as a germ cell marker to identify a germline because vasa RNA is a component of germ plasm. In this study, we described the expression pattern of black rockfish (Sebastes schlegelii) vasa (Ssvas) in gonadal formation and development by in situ hybridization. The results showed that Ssvas failed in localization at the cleavage furrows until the late gastrula stage, when PGCs appeared and migrated to the genital ridge and formed elongated gonadal primordia at 10 days after birth. This study firstly revealed the PGCs origination and migration characteristics in viviparous marine fish. Furthermore, we microinjected chimeric mRNA containing EGFP and the 3′untranslated region (3′UTR) of Ssvas into zebrafish (Danio rerio) and marine medaka (Oryzias melastigma) fertilized eggs for tracing PGCs. We found that, although Sebastes schlegelii lacked early localization, similar to red seabream (Pagrus major) and marine medaka, only the 3′UTR of Ssvas vasa 3′UTR of black rockfish was able to label both zebrafish and marine medaka PGCs. In comparison with other three Euteleostei species, besides some basal motifs, black rockfish had three specific motifs of M10, M12, and M19 just presented in zebrafish, which might play an important role in labeling zebrafish PGCs. These results will promote germ cell manipulation technology development and facilitate artificial reproduction regulation in aquaculture.

Karlodinium elegans sp. nov. (Gymnodiniales, Dinophyceae), a novel species isolated from the East China Sea in a dinoflagellate bloom

On May 24–29, 2019, a harmful algal bloom occurred in Pingtan coastal areas, Fujian, southeast China, and caused mass mortality of cage-cultured fish. Two clonal cultures of an unknown naked dinoflagellate were set up from seawater samples taken during the bloom. The cultures were examined for morphological features, ultrastructure characters, photosynthetic pigments, the large subunit (LSU) of the rRNA gene, and the internal transcribed spacer (ITS) sequences, as well as acute toxicity analysis. The cell was unarmored, small-sized, and ovoid, and was characterized by elaborate striations on the epicone and hypocone. The nucleus was large, ellipsoid to oval or kidney-shaped, and centrally located in the cell. A long linear apical groove originated above the sulcus in the ventral epicone and extended to the dorsal side. An elongate, slit-like “ventral pore” was located on the left of the epicone, well away from the apical groove. The chloroplasts were yellowish brown, numerous, band like, and irregularly distributed in the cell periphery. Fucoxanthin was the main accessory pigment composition. Phylogeny topology reconstructed on partial LSU rDNA showed that the unknown dinoflagellate branched as a sister species to Karlodinium sp. (strain IFR981 & IFR797, from France) and Karlodinium corrugatum (strain KDGSO08, from Australia), with genetic divergences of 0.6% and 3.3%, respectively. Based on the morphological and molecular phylogenetic analysis, we describe the novel dinoflagellate as Karlodinium elegans sp. nov. A toxicity assay revealed that the clonal culture of K. elegans (strain PTB601) had no adverse effect on brine shrimp (Artemia salina) and marine medaka (Oryzias melastigma), indicating it may not be a toxic species.

Microplastics decrease the toxicity of triphenyl phosphate (TPhP) in the marine medaka (Oryzias melastigma) larvae

Plastics have been recognized as a serious threat to the environment. Besides their own toxicity, microplastics can interact with other environmental pollutants, acting as carriers and potentially modulating their toxicity. In this study, the toxicity of polystyrene (PS) microplastic fragments (plain PS; carboxylated PS, PS-COOH and aminated PS, PS-NH2) and triphenyl phosphate (TPhP) (an emerging organophosphate flame retardant) at the environmentally relevant concentrations to the marine medaka (Oryzias melastigma) larvae was investigated. Larvae were exposed to 20 μg/L of microplastic fragments or 20 and 100 μg/L of TPhP or a combination of both for 7 days. The results showed that the three microplastics did not affect the larval locomotor activity. For TPhP, the larval moving duration and distance moved were significantly decreased by the TPhP exposure, with a maximum decrease of 43.5% and 59.4% respectively. Exposure to 100 μg/L TPhP respectively down-regulated the expression levels of sine oculis homeobox homologue 3 (six3) and short wavelength-sensitive type 2 (sws2) by 19.1% and 41.7%, suggesting that TPhP might disturb eye development and photoreception and consequently the low locomotor activity in the larvae. Interestingly, during the binary mixture exposure, the presence of PS, PS-COOH or PS-NH2 reversed the low locomotor activity induced by 100 μg/L TPhP to the normal level. Relative to the larvae from the 100 μg/L TPhP group, the movement duration and distance moved were increased by approximately 60% and 100%, respectively, in the larvae from the TPhP + PS, TPhP + PS-COOH and TPhP + PS-NH2 groups. However, the gene expression profiles were distinct among the fish from the TPhP + PS, TPhP + PS-COOH and TPhP + PS-NH2 groups, implying different mechanisms underlying the reversal of the locomotor activity. The findings in this study challenge the general view that microplastics aggravate the toxicity of the adsorbed pollutants, and help better understand the environmental risk of microplastic pollution.