Marine Medaka Oryzias Melastigma Research Articles

Secondary PVC microplastics are more toxic than primary PVC microplastics to Oryzias melastigma embryos

Irregular-shaped and partially degraded secondary microplastics (SMP) account for the majority of MPs in marine environments, yet little is known about their effects on marine organisms. In this study, we investigated the embryotoxicity of polyvinyl chloride SMP and primary microplastics (PMP) to the marine medaka Oryzias melastigma. This study aimed to determine the physical impacts of MPs and, for the first time, elucidate the underlying mechanisms of physical toxicity. SMP shortened hatching time and induced higher teratogenic effects on larvae relative to PMP, indicating a higher toxicity from SMP. Physical damage from SMP to the chorion surface appears to be the main toxicity mechanism, caused by their irregular shape and reduced aggregation relative to PMP. In contrast, real-time changes in oxygen demonstrated that hypoxia caused by greater PMP adsorption to the chorion surface contributes to the toxicological responses of this material relative to SMP. Modulation of genes involved in hypoxia-response, cardiac development and hatching confirmed the toxicity mechanisms of PMP and SMP. The chemical contribution to observed toxicity was negligible, confirming impacts derived from physical toxicity. Our findings highlight the negative effects of environmentally relevant SMP on the marine ecosystems.

Subchronic toxicity of dietary sulfamethazine and nanoplastics in marine medaka (Oryzias melastigma): Insights from the gut microbiota and intestinal oxidative status

Antibiotics and nanoplastics are two prevalent pollutants in oceans, posing a great threat to marine ecosystems. As antibiotics and nanoplastics are highly bioconcentrated in lower trophic levels, evaluating their impacts on marine organisms via dietary exposure route is of great importance. In this study, the individual and joint effects of dietborne sulfamethazine (SMZ) and nanoplastic fragments (polystyrene, PS) in marine medaka (Oryzias melastigma) were investigated. After 30 days of dietary exposure, 4.62 mg/g SMZ decreased the Chao1 index (60.86% for females and 26.85% for males) and the Shannon index (68.95% for females and 65.05% for males) and significantly altered the structure of gut microbial communities in both sexes. The female fish exposed to 4.62 mg/g SMZ exhibited higher intestinal sod (43.5%), cat (38.5%) and gpx (39.6%) transcripts, indicating oxidative stress in the gut. PS alone at 3.45 mg/g slightly altered the composition of the gut microbiota. Interestingly, the mixture of SMZ and PS caused more modest effects on the gut microbiota and intestinal antioxidant physiology than the SMZ alone, suggesting that the presence of PS might alleviate the intestinal toxicity of SMZ in a scenario of dietary co-exposure. This study helps better understand the risk of antibiotics and nanoplastics to marine ecosystems.

Norethisterone exposure alters the transcriptome of Marine Medaka (Oryzias melastigma) larvae

ABSTRACT Norethisterone (NET) is one of the earliest synthetic progestins. Its widespread and long-term use means that NET is often detected on the surface and in groundwater samples, which may cause harm to aquatic organisms. Marine medaka (Oryzias melastigma) is a brackish fish, often used in ecotoxicology studies. In this study, the effect of norethisterone exposure on the transcriptome of marine medaka larvae was analyzed. Larvae were exposed to 10 ng/L NET for 24 h, then RNA-Seq was performed. Exposure to 10 ng/L NET resulted in 306 up-regulated and 516 down-regulated genes. An enrichment analysis revealed that differentially expressed genes (DEGs) were significantly enriched in metabolic and biosynthetic pathways, including DNA replication, proteasome, steroid biosynthesis, and other amino acid metabolism pathways. In the DNA replication pathway, all the DEGs were down-regulated, while in the steroid biosynthesis pathway, sqle, hmgcr, dhcr7, and dhcr24 were down-regulated. Quantitative real-time PCR (qPCR) was used to verify 12 DEGs, confirming that the validity of the RNA-Seq analysis. These results show that even low environmental concentrations of NET can lead to transcription level changes of genes related to fish growth and development, which may have adverse effects on the early development and growth of aquatic organisms. Highlights Transcriptomes from norethisterone-exposed marine medaka larvae were obtained A total of 822 differentially expressed genes were identified Norethisterone affected 155 pathways, including DNA replication.

Polystyrene microplastics increase estrogenic effects of 17α-ethynylestradiol on male marine medaka (Oryzias melastigma)

Microplastics (MPs) and endocrine disrupting chemicals are ubiquitous pollutants in marine environments, but their combined ecological risk is unclear. This study exposed male marine medaka (Oryzias melastigma) to 10 ng/L 17α-ethynylestradiol (EE2) alone or EE2 plus 2, 20, and 200 μg/L polystyrene MPs for 28 days to investigate the impacts of MPs on the reproductive disruption of EE2. The results showed that 10 ng/L EE2 alone did not affect biometric parameters, while co-exposure to EE2 and 20, 200 μg/L MPs suppressed the growth and decreased gonadosomatic and hepatosomatic indices. Compared to EE2 alone, EE2 plus MPs exposure significantly increased plasma 17β-estradiol (E2) levels in a dose-dependent manner, and co-exposure to EE2 and 20, 200 μg/L MPs significantly increased the ratios of E2/testosterone (T). Moreover, EE2 plus MPs exposure elevated the transcription levels of estrogen biomarker genes vitellogenin and choriogenin, and estrogen receptor (ERα and ERβ). Morphological analysis also showed that co-exposure to EE2 and MPs induced more severe damage to the testes and livers, indicating that MPs increased the toxicity of EE2. The actual EE2 concentrations in the solution increased with the exposure concentrations of MPs, suggesting that MPs changed the fate and behavior of EE2 in the seawater. These findings demonstrate that MPs could increase the estrogenic effects of EE2 on marine fish, suggesting that the combined health risk of MPs and endocrine disrupting chemicals on marine organisms should be paid great attention.

Chronic exposure to environmentally realistic levels of diuron impacts the behaviour of adult marine medaka (Oryzias melastigma).

Diuron, a commonly used herbicide and antifouling biocide, has been frequently detected in seawater. The effects of diuron on fish behaviour are currently poorly understood. Herein, the marine medaka (Oryzias melastigma) was continuously exposed to environmentally realistic levels of diuron from the fertilised egg stage to the adult stage. Behavioural evaluation of adult marine medaka indicated that exposure to diuron increased anxiety in the light-dark test and increased predator avoidance. In addition, diuron exposure significantly reduced aggression, social interaction, shoaling, and learning and memory ability. However, only negligible variations in foraging behaviour and in behaviour in the novel tank test were observed. Marine medaka chronically exposed to diuron also showed decreased levels of dopamine in the brain, and changes were observed in the transcription of genes related to dopamine synthesis, degradation and receptors. Exposure to 5000 ng/L diuron caused significant downregulation of the expression of the genes of tyrosine hydroxylase and monoamine oxidase and significantly upregulated the expression of the genes of the D5 dopaminergic receptor. The relative expression of the D4 dopaminergic receptor was significantly upregulated in the 50, 500 and 5000 ng/L diuron-treated groups. These findings highlight the significant neurotoxic effects of diuron and the extent to which this may involve the dopaminergic system of the brain. More broadly, this study reveals the ecological risk associated with environmentally realistic levels of diuron in marine animals.

Parental exposure to sulfamethazine and nanoplastics alters the gut microbial communities in the offspring of marine madaka (Oryzias melastigma)

The individual and combined toxicity of antibiotics and nanoplastics in marine organisms has received increasing attention. However, many studies have been mostly focused on the impacts on the directly exposed generation (F0). In this study, intergenerational effects of sulfamethazine (SMZ) and nanoplastic fragments (polystyrene, PS) on the growth and the gut microbiota of marine medaka (Oryzias melastigma) were investigated. The results showed that parental exposure to dietary SMZ (4.62 mg/g) alone and PS (3.45 mg/g) alone for 30 days decreased the body weight (by 13.41% and 34.33%, respectively) and altered the composition of gut microbiota in F1 males (two months after hatching). Interestingly, parental exposure to the mixture of SMZ and PS caused a more modest decrease in the body weight of F1 males than the PS alone (15.60% vs 34.33%). The hepatic igf1 level and the relative abundance of the host energy metabolism related phylum Bacteroidetes for the SMZ + PS group were significantly higher than those for the PS group (igf1, increased by 97.1%; Bacteroidetes, 2.876% vs 0.375%), suggesting that the parentally derived mixture of SMZ and PS might influence the first microbial colonization of gut in a different way to the PS alone. This study contributes to a better understanding of the long-term risk of antibiotics and nanoplastics to marine organisms.

Effect of Modified Clay on the Growth Dynamics and Physio—Biochemical Response of Newly Hatched Larvae of the Marine Medaka (Oryzias melastigma)

An outbreak of harmful algal blooms (HABs) often leads to the death of fish and other marine organisms and causes serious losses to human economic activities. Modified clay (MC) technology is an effective way to control HABs. Although the material and preparation process are based on the premise of green and safety, the potential eco-environmental impacts of MC on non-target organisms should still have to receive attention before field applications can occur. Therefore, the effects of one commonly used modified clay, polyaluminum chloride-modified clay (PAC-MC), on the survival, growth, and oxidative stress of the marine medaka (Oryzias melastigma) were studied. The toxicity test results showed that the 96-h median lethal concentration (96-h LC50) of PAC-MC for newly hatched medaka larvae was 5.204 g/L, which was much higher than the concentration used on site (4–10 t/km2). Within the concentration range of PAC-MC used in this experiment (≤2 g/L), the morphology, heart rate, growth, and aluminum content of larvae did not change with the increase in the modified clay concentration. Low concentrations of PAC-MC (≤0.5 g/L) did not significantly affect catalase (CAT) activity, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and the content of malondialdehyde (MDA), but higher concentrations of PAC-MC (such as 2 g/L) caused oxidative damage to the larvae and increased the antioxidant enzyme activity of the larvae. The present study revealed that under an effective dosage for treating harmful algal blooms on site, PAC-MC had no adverse effects on the survival, growth, oxidative stress, and aluminum content of the newly hatched marine medaka, which provides a scientific basis for the field application of modified clay.

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.

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.

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.

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.