Comparative biochemical characterization of pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) homologs in the plasma from four Takifugu species: Conservation of heat-stable PSTBP orthologs having three and two tandemly repeated lipocalin domains in genus Takifugu.

To study the relationship between domain characteristics of pufferfish saxitoxin and tetrodotoxin binding protein (PSTBP) proteoforms and their thermal stability, a comparative biochemical characterization of PSTBPs from the plasma of four Takifugu species (T. flavipterus, T. pardalis, T. alboplumbeus and T. rubripes) was conducted by Western blot analysis. The heat-tolerance tetrodotoxin (TTX)-binding ability of PSTBP proteoforms in T. rubripes plasma was verified by ultrafiltration and liquid chromatography tandem mass spectrometry (LC-MS/MS). These results suggest that the heat-stable PSTBP proteoforms, composed of three and two tandemly repeated lipocalin domains, are genetically conserved and ubiquitous in the genus Takifugu. This study builds on our knowledge of the structural and functional properties of PSTBP proteoforms, which is vital for understanding how toxins are transmitted and accumulate in organisms and is essential for evaluating the potential risks of toxins in seafood.

Diurnal-Rhythmic Relationships between Physiological Parameters and Photosynthesis- and Antioxidant-Enzyme Genes Expression in the Raphidophyte Chattonella marina Complex.

Diurnal rhythms in physiological functions contribute to homeostasis in many organisms. Although relationships between molecular biology and diurnal rhythms have been well studied in model organisms like higher plants, those in harmful algal bloom species are poorly understood. Here we measured several physiological parameters and the expression patterns of photosynthesis-related and antioxidant-enzyme genes in the Chattonella marina complex to understand the biological meaning of diurnal rhythm. Under a light-dark cycle, Fv/Fm and expression of psbA, psbD, and 2-Cys prx showed significant increases in the light and decreases during the dark. These rhythms remained even under continuous dark conditions. DCMU suppressed the induction of psbA, psbD, and 2-Cys prx expression under both light regimes. Oxidative stress levels and H2O2 scavenging activities were relatively stable, and there was no significant correlation between H2O2 scavenging activities and antioxidant-enzyme gene expression. These results indicate that the Chattonella marina complex has developed mechanisms for efficient photosynthetic energy production in the light. Our results showed that this species has a diurnal rhythm and a biological clock. These phenomena are thought to contribute to the efficiency of physiological activities centered on photosynthesis and cell growth related to the diurnal vertical movement of this species.

Combined effect of anthracene and polyethylene microplastics on swimming speed and cytochrome P4501A monooxygenase expression of Java medaka (Oryzias javanicus).

Microplastics have been detected in a variety of aquatic ecosystems, and the combined effect of microplastics and chemical pollutants has become a matter of increasing concern. We conducted a 12-d co-exposure test of anthracene and spherical or fragmented polyethylene microplastics (size 200 µm) on Java medaka (Oryzias javanicus). The accumulation of anthracene in Java medaka muscle reached a plateau on day 5 in all anthracene exposure groups, and no significant differences were detected among the groups (ANT, 20.4 ± 5.5; ANT + SPPE-MP, 24.7 ± 2.7; ANT + FRPE-MP, 24.6 ± 4.7 µg/g). However, co-exposure to anthracene and spherical or fragmented polyethylene microplastics increased the duration of slow swimming in a swimming behavior test (control, 4.1 ± 1.4; ANT, 5.2 ± 2.8; ANT + SPPE-MP, 12.4 ± 3.7; ANT + FRPE-MP, 17.4 ± 5.1 min/30 min), and co-exposure to anthracene and fragmented polyethylene microplastics induced higher cytochrome P4501A monooxygenase (CYP1A) expression in Java medaka livers than the other anthracene exposure groups (ANT, 189 ± 74; ANT + SPPE-MP, 203 ± 75; ANT + FRPE-MP 272 ± 36% of control). Polyethylene microplastics appear to be weak vectors of anthracene at the size tested (200 µm), and the effect of shape (spherical or fragmented) on the vector effect was small. However, the presence of polyethylene microplastics could affect the swimming behavior and CYP1A expression in Java medaka.

Size effect of polystyrene microplastics on the accumulation of anthracene for Java medaka (Oryzias javanicus).

Pollution by microplastics in aquatic ecosystems is a worldwide problem, and the role of microplastics as vectors of pollutants has been a concern. Although small microplastics are thought to have a greater effect than large microplastics as vectors of pollutants, the impact of the size of microplastics on their ability to serve as vectors of pollutants has not been quantified. In this study, we conducted the 14-day experiment (7 days of exposure and 7 days of depuration) with polystyrene microplastics (2-µm or 10-µm diameter) and anthracene. On the last day of the exposure period, the concentration of anthracene in the muscle of Java medaka exposed to both anthracene and 2-µm polystyrene microplastics was the highest (47.4 ± 15.2 µg/g-muscle) of any group, followed by the group exposed to both anthracene and 10-µm polystyrene microplastics (23.0 ± 4.2 µg/g-muscle) and the group exposed to only anthracene (11.2 ± 2.2 µg/g-muscle). These results demonstrated that the size of microplastics was a critical determinant of their ability to serve as vectors of anthracene. The concentrations of anthracene and fine microplastics in the environment are sufficiently low that the effect of microplastics as vectors of anthracene may be observed only under experimental conditions that are unlikely to occur in the present environment. However, because pollution by plastics is expected to become more serious in the future, careful thought and proactive action will be needed to ensure that the impact of microplastics as vectors of pollutants does not become demonstrable under future environmental conditions.

Peek-A-Boo Test: A Simple Test for Assessing the Effect of Anxiolytics on Fish Behavior.

The potential of pharmaceuticals and personal care products to alter the behavior of aquatic organisms is a growing concern. To assess the actual effect of these substances on aquatic organisms, a simple but effective behavioral test is required. We devised a simple behavioral (Peek-A-Boo) test to assess the effect of anxiolytics on the behavior of a model fish (medaka, Oryzias latipes). In the Peek-A-Boo test, we investigated the response of medaka to an image of a predator fish (donko fish, Odontobutis obscura). The test revealed that the time taken for test medaka exposed to diazepam (0.8, 4, 20, or 100 µg/L) to approach the image was shorter by a factor of 0.22 to 0.65, and the time spent in the area close to the image was longer by a factor of 1.8 to 2.7 than in the solvent control group for all diazepam exposure groups (p < 0.05). Hence, we confirmed that the test could detect changes in medaka behavior caused by diazepam with high sensitivity. The Peek-A-Boo test we devised is a simple behavioral test with high sensitivity for fish behavioral alteration. Environ Toxicol Chem 2023;42:2358-2363. © 2023 SETAC.

Differences in microplastic degradation in the atmosphere and coastal water environment from two island nations: Japan and New Zealand.

Microplastics are subject to environmental forces that can change polymer organization on a molecular scale. However, it is not clear to what extent these changes occur in the environment and whether microplastics in the atmospheric and water environment differ. Here we identify structural differences between microplastics in the atmosphere and water environment from Japan and New Zealand, representing two archipelagos differing in their proximity to nearby countries and highly populated areas. We first highlight the propensity for smaller microplastics to arrive via air masses from the Asian continent to the Japan Sea coastal area, while New Zealand received larger, locally derived microplastics. Analyses of polyethylene in the Japanese atmosphere indicate that microplastics transported to the Japanese coastal areas were more crystalline than polyethylene particles in the water, suggesting that the plastics arriving by air were relatively more aged and brittle. By contrast, polypropylene particles in New Zealand waters were more degraded than the microplastic particles in the air. Due to the lack of abundance, both polyethylene and polypropylene could not be analyzed for both countries. Nevertheless, these findings show the structural variation in microplastics between environments in markedly different real-world locations, with implications for the toxic potential of these particles.

Tributyltin-binding protein type 1 (fish acid glycoprotein) is a potential gatekeeper of ethinylestradiol action in fish.

Tributyltin (TBT)-binding protein type 1 in Japanese medaka (Oryzias latipes) (O.latTBT-bp1) is a fish lipocalin implicated in TBT binding and detoxification. We purified recombinant O.latTBT-bp1 (rO.latTBT-bp1; ca. 30 kDa) by using a baculovirus expression system and His- and Strep-tag chromatography process. Then, we examined O.latTBT-bp1 binding to several endo/exogenous steroid hormones by means of competitive binding assay. The dissociation constants for the binding of rO.latTBT-bp1 to DAUDA and ANS, two fluorescent ligands of lipocalin, were 7.06 and 13.6 µM, respectively. Multiple model validations indicated that a single-binding-site model was the most appropriate for evaluating rO.latTBT-bp1 binding. In the competitive binding assay, testosterone, 11-ketotestosterone, and 17ß-estradiol were each bound by rO.latTBT-bp1; rO.latTBT-bp1 showed the strongest affinity for testosterone (inhibition constant, Ki = 3.47 µM). Endocrine-disrupting chemical (synthetic steroid) also bound to rO.latTBT-bp1; the affinity for ethinylestradiol (Ki = 9.29 µM) was stronger than that for 17ß-estradiol (Ki = 30.0 µM). To determine the function of O.latTBT-bp1, we produced TBT-bp1 knockout medaka (TBT-bp1 KO), which we exposed to ethinylestradiol for 28 days. After exposure, the number of papillary processes in TBT-bp1 KO genotypic male medaka was significantly fewer (3.5), compared to that in wild-type male medaka (22). Thus, TBT-bp1 KO medaka were more sensitive to the anti-androgenic effects of ethinylestradiol than wild-type medaka. These results indicate that O.latTBT-bp1 may bind to steroids and act as a gatekeeper of ethinylestradiol action by regulating the androgen-estrogen balance.

Effects of the antifouling agent tributyltin on the sinking behavior, photosynthetic rate and biochemical composition of the marine planktonic diatom Thalassiosira pseudonana.

This study investigated the changes in the sinking rates and physiochemical characteristics of the planktonic marine diatom, Thalassiosira pseudonana, caused by 72 h exposure to antifouling agent tributyltin (TBT) at 1.0 µg L-1 (72-h 10% effective concentration for growth rate, EC10), and 1.7 µg L-1 (EC50). After 72 h of exposure, the sinking rates of T. pseudonana cells were changed from 0.13-0.08 m day-1 in the control, 0.08-0.05 m day-1 in the EC10 treatment, and 0.04-0.006 m day-1 in the EC50 treatment. The results revealed that the sinking rate of T. pseudonana decreased significantly compared with the control at 48 h in the EC10 treatment group and at 24, 48, and 72 h in the EC50 treatment group. The photosynthetic performance index on an absorption basis and the maximum quantum yields of photosystem II also decreased significantly (P < 0.05) in the TBT treatments compared with the control. There was a significant (P < 0.05) positive correlation between sinking rates and cellular protein contents (ng cell-1). Changes in the biochemical and physiochemical composition of the cells suggest that interference with photosynthetic processes by TBT may have reduced their specific gravity and thereby caused a decrease in the sinking rates of T. pseudonana. The results of this investigation suggest the importance of considering the effects of pollutants on the sinking behaviors of diatoms when evaluating the adverse effects of pollutants on marine primary production.

Hydroxylated benzo[c]phenanthrene metabolites cause osteoblast apoptosis and skeletal abnormalities in fish.

To study the toxicity of 3-hydroxybenzo[c]phenanthrene (3-OHBcP), a metabolite of benzo[c]phenanthrene (BcP), first we compared it with its parent compound, BcP, using an in ovo-nanoinjection method in Japanese medaka. Second, we examined the influence of 3-OHBcP on bone metabolism using goldfish. Third, the detailed mechanism of 3-OHBcP on bone metabolism was investigated using zebrafish and goldfish. The LC50s of BcP and 3-OHBcP in Japanese medaka were 5.7 nM and 0.003 nM, respectively, indicating that the metabolite was more than 1900 times as toxic as the parent compound. In addition, nanoinjected 3-OHBcP (0.001 nM) induced skeletal abnormalities. Therefore, fish scales with both osteoblasts and osteoclasts on the calcified bone matrix were examined to investigate the mechanisms of 3-OHBcP toxicity on bone metabolism. We found that scale regeneration in the BcP-injected goldfish was significantly inhibited as compared with that in control goldfish. Furthermore, 3-OHBcP was detected in the bile of BcP-injected goldfish, indicating that 3-OHBcP metabolized from BcP inhibited scale regeneration. Subsequently, the toxicity of BcP and 3-OHBcP to osteoblasts was examined using an in vitro assay with regenerating scales. The osteoblastic activity in the 3-OHBcP (10-10 to 10-7 M)-treated scales was significantly suppressed, while BcP (10-11 to 10-7 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10-11 to 10-7 M). The detailed toxicity of 3-OHBcP (10-9 M) in osteoblasts was then examined using gene expression analysis on a global scale with fish scales. Eight genes, including APAF1, CHEK2, and FOS, which are associated with apoptosis, were identified from the upregulated genes. This indicated that 3-OHBcP treatment induced apoptosis in fish scales. In situ detection of cell death by TUNEL methods was supported by gene expression analysis. This study is the first to demonstrate that 3-OHBcP, a metabolite of BcP, has greater toxicity than the parent compound, BcP.

Combined effect of diazepam and polystyrene microplastics on the social behavior of medaka (Oryzias latipes).

The combined effect of microplastics and pharmaceuticals on aquatic organisms is an issue of concern. In this laboratory study, we evaluated the combined effect of polystyrene microplastics (2-µm diameter) and diazepam on the social behavior of medaka (Oryzias latipes) by using the shoaling behavior test with five treatment groups: solvent control, polystyrene microplastics exposure (0.04 mg/L), low-concentration diazepam exposure (0.03 mg/L), high-concentration diazepam exposure (0.3 mg/L), and polystyrene microplastics and low-concentration diazepam co-exposure. After 7 days of exposure, the shoal-leaving behavior of the high-concentration diazepam exposure group (8.9 ± 8.3 counts/medaka) and the co-exposure group (6.8 ± 6.7 counts/medaka) was significantly greater than that in the solvent control group (1.8 ± 2.6 counts/medaka). Even after 5 days of recovery, medaka in the co-exposure group left the shoal more often (7.3 ± 5.0 counts/medaka) than those in the solvent control group (2.6 ± 2.6 counts/medaka), whereas the shoal-leaving behavior in other exposure groups, except for the high-concentration diazepam exposure group, was restored. Our findings show that the combined effects of diazepam and polystyrene microplastics suppressed medaka social behavior, suggesting that the presence of microplastics can enhance the adverse effects of pollutants on the social behavior of aquatic organisms.

Persistent impact of amitriptyline on the behavior, brain neurotransmitter, and transcriptional profile of zebrafish (Danio rerio).

Discontinuation of amitriptyline (AMI) has been demonstrated to induce long-term withdrawal syndromes in mammals. However, no studies have focused on the persistent impacts of short-term AMI exposure on teleosts. Here, following exposure to AMI (2.5 and 40 µg/L) for 7 days (E7), zebrafish were transferred into AMI-free water to recover for 21 days (R21). The behavior, brain neurotransmitters, and brain transcriptional profiles were investigated on E7 and R21. AMI exposure induced persistent hypoactivity (2.5 and 40 µg/L) and abnormal schooling behavior (40 µg/L). AMI also induced long-term impacts on the brain serotonin (5-HT), 5-hydroxyindoleacetic acid, norepinephrine, and acetylcholine levels, several of which showed significant correlations with the locomotor activity or schooling behavior. Transcriptional analysis revealed persistent dysregulation in the pathways involved in the circadian rhythm, glycan biosynthesis and metabolism, and axon guidance in brain samples. Twelve genes were predicted as key driver genes in response to AMI exposure, and their significantly differential expression may direct changes across the related molecular networks. Moreover, upregulated brain 5-HT may serve as the central modulator of the persistent AMI pathogenesis in zebrafish. Considering AMI residues in natural waters may temporarily exceed µg/L, corresponding persistent adverse effects on teleosts should not be ignored.

Obesogenic and developmental effects of TBT on the gene expression of juvenile Japanese medaka (Oryzias latipes).

The widely used antifoulant tributyltin chloride (TBT) is highly toxic to aquatic organisms. In the present study, four-week-old Japanese medaka (Oryzias latipes) juveniles were orally exposed to TBT at 1 and 10 ng/g bw/d for 1, 2, and 4 weeks, respectively. Half of the tested medaka juveniles showed bone morphology alterations in both 1 and 10 ng/g bw/d TBT 4-week exposure groups. Nile Red (NR) staining showed that the juveniles exposed to 1 ng/g bw/d TBT for 2 and 4 weeks had significantly enlarged adipocyte areas. The mRNA-Seq analysis indicated that 1 ng/g bw/d TBT exposure for 2 weeks affected bone morphology through developmental processes. The GO and KEGG analyses suggested that the adipogenic effect of TBT observed in this study may be induced by metabolic processes, oxidative phosphorylation, and fatty acid degradation and metabolism pathways. Therefore, both morphological observation and mRNA-Seq analysis showed obesogenic effects and developmental toxicity of TBT to juvenile Japanese medaka.

Uptake and depuration kinetics of microplastics with different polymer types and particle sizes in Japanese medaka (Oryzias latipes).

Microplastic (MP) pollution and the related impacts on aquatic species have drawn worldwide attention. However, knowledge of the kinetic profiles of MPs in fish remains fragmentary. In this study, we conducted exposure and depuration tests of the following fluorescent-labeled MPs: polyethylene (PE; sphere with 200 or 20 µm diameter) and polystyrene (PS; sphere with 20 or 2 µm diameter) using juvenile Japanese medaka (Oryzias latipes). The distribution and concentration of MPs in medaka were directly determined in-situ after tissue transparency. During the 14-day exposure, MPs was mainly detected in the gastrointestinal tract, while some MPs at the size of ≤ 20 µm were located in the area of the gills and head. The bioconcentration factor (BCF; L/kg) for MPs in medaka was estimated as 74.4 (200 µm PE), 25.7 (20 µm PE), 16.8 (20 µm PS), and 139.9 (2 µm PS). Within the first five days of depuration, MPs were exponentially eliminated from the fish body, but 2 µm PS-MPs could be still detected in the gastrointestinal tract at the end of the 10-day depuration phase. Our results suggest that MPs 2 µm in diameter may pose ecological risks to aquatic species due to their relatively higher BCF and the potential for long-term persistence in the body.

Recent Progress in the Study of Peroxiredoxin in the Harmful Algal Bloom Species Chattonella marina.

Peroxiredoxin (Prx) is a relatively recently discovered antioxidant enzyme family that scavenges peroxides and is known to be present in organisms from biological taxa ranging from bacteria to multicellular eukaryotes, including photosynthetic organisms. Although there have been many studies of the Prx family in higher plants, green algae, and cyanobacteria, few studies have concerned raphidophytes and dinoflagellates, which are among the eukaryotic algae that cause harmful algal blooms (HABs). In our proteomic study using 2-D electrophoresis, we found a highly expressed 2-Cys peroxiredoxin (2-CysPrx) in the raphidophyte Chattonella marina var. antiqua, a species that induces mass mortality of aquacultured fish. The abundance of the C. marina 2-CysPrx enzyme was highest in the exponential growth phase, during which photosynthetic activity was high, and it then decreased by about a factor of two during the late stationary growth phase. This pattern suggested that 2-CysPrx is a key enzyme involved in the maintenance of high photosynthesis activity. In addition, the fact that the depression of photosynthesis by excessively high irradiance was more severe in the 2-CysPrx low-expression strain (wild type) than in the normal-expression strain (wild type) of C. marina suggested that 2-CysPrx played a critical role in protecting the cell from oxidative stress caused by exposure to excessively high irradiance. In the field of HAB research, estimates of growth potential have been desired to predict the population dynamics of HABs for mitigating damage to fisheries. Therefore, omics approaches have recently begun to be applied to elucidate the physiology of the growth of HAB species. In this review, we describe the progress we have made using a molecular physiological approach to identify the roles of 2-CysPrx and other antioxidant enzymes in mitigating environmental stress associated with strong light and high temperatures and resultant oxidative stress. We also describe results of a survey of expressed Prx genes and their growth-phase-dependent behavior in C. marina using RNA-seq analysis. Finally, we speculate about the function of these genes and the ecological significance of 2-CysPrx, such as its involvement in circadian rhythms and the toxicity of C. marina to fish.

Quantifying the vector effects of polyethylene microplastics on the accumulation of anthracene to Japanese medaka (Oryzias latipes).

To better assess the risk of microplastics (MPs) as a vector for contaminants, it is essential to understand the relative importance of MPs compared to other pathways for chemical transfer as well as the consequences of co-exposure. In this study, we exposed Japanese medaka (Oryzias latipes) to anthracene (ANT, 0.1 mg/L) in the presence or absence of pristine polyethylene MPs (PE-MPs, 106 beads/L), to quantify the vector effect of PE-MPs on ANT accumulation. Under the ANT-MPs co-exposure conditions, PE-MPs rapidly accumulated in the gastrointestinal tract of the medaka during a 14-day uptake phase, with an average bioconcentration factor of 171.4 L/kg. The PE-MPs could absorb and accumulate approximately 70 % of the ANT from the water sample. The PE-MPs changed the pharmacokinetic profile of ANT in medaka by decreasing both the uptake and depuration rate constants. The one compartment with first-order elimination model estimated that the amounts of ANT in the water phase and absorbed by PE-MPs (i.e., a vector effect) contributed about 67 % and 33 % of the ANT accumulation in medaka, respectively. At the end of the uptake (exposure) phase, however, the presence of PE-MPs did not significantly alter the final ANT concentrations in the fish body or alter the behavioral impacts of ANT. Thus, PE-MPs ingestion may act as a vector to concentrate and transfer ANT to medaka, but the presence of these particles may have limited adverse effects on fish under co-exposure systems of the type used in this study.

Short-term and persistent impacts of sublethal exposure to diazepam on behavioral traits and brain GABA levels in juvenile zebrafish (Danio rerio).

Environmental pollution by the psychoactive drug diazepam (DZP) has been suggested to disrupt various behavioral traits of fishes. Exposure to DZP in natural waters may be of episodic duration, but there are few reports on the persistence of abnormal behaviors of fishes caused by such acute exposure. In the current study, we exposed juvenile zebrafish (Danio rerio) to sublethal doses of DZP (1200, 120, and 12 µg/L) for four days and evaluated their behavioral traits and brain γ-aminobutyric acid (GABA) levels at days 0 (i.e., immediately after the 4-day exposure), 7, and 21 of the recovery period. Exposure to DZP induced short-term impairment of swimming ability and two-fish interactions of zebrafish. In contrast, DZP induced persistent and/or delayed effects on locomotor activity of zebrafish, i.e., hypoactivity at 1200 µg/L and hyperactivity at 120 and 12 µg/L, that could be still observed on days 7 and/or 21 during the recovery period. DZP exposure also exhibited concentration-specific effects on brain GABA levels in zebrafish, i.e., decreased at 1200 µg/L and increased at 120 and 12 µg/L. Correlation analysis suggested that the changes in brain GABA levels may contribute to the persistence of abnormalities in the locomotor activity of zebrafish. Our findings suggest that zebrafish need a long time to recover from acute exposure to DZP, thus highlighting that the persistence of behavioral abnormalities induced by such psychoactive drugs should be considered in order to better assess their risks in natural ecosystems.

Production of a tributyltin-binding protein 2 knockout mutant strain of Japanese medaka, Oryzias latipes.

Tributyltin-binding proteins (TBT-bps), members of the lipocalin family, bind TBT in fish blood and are presumed to contribute to detoxification of TBT. Recent studies have shown that many fish species have TBT-bp genes, and that these genes are induced by stresses such as exposure to chemicals or fish pathogenic bacteria. However, the function of TBT-bps, and the mechanisms of their induction and detoxification activity are still unclear. Here, towards elucidating the functions of TBT-bp2, we produced a TBT-bp2 knockout (TBT-bp2-/-) strain of Japanese medaka, Oryzias latipes, by using the CRISPR/Cas9 system. Gene expression of the mutated TBT-bp2 was reduced, and the cDNA sequencing and predicted protein structure suggested possible loss of function. However, the fish could be grown under normal conditions. Exposure of the TBT-bp2-/- strain of medaka to various stresses in future experiments is expected to contribute to our understanding of this novel detoxification system in aquatic organisms.

Detoxification roles of tributyltin-binding protein type 2 in Japanese medaka (Oryzias latipes) exposed to tributyltin.

Tributyltin-binding protein type 2 (TBT-bp2), a homolog of α1-acid glycoprotein, may contribute to both accumulation and detoxification of TBT in fish. In this study, we conducted acute TBT exposure tests using both wide-type (WT) and TBT-bp2-/- (KO) strains of medaka and compared their responses in survival time and accumulation of TBT. Deficiency of TBT-bp2 significantly accelerated the time to death of medaka and decreased the LC50 of TBT, indicating that the KO-strain is more sensitive to TBT. No significant difference in the intrinsic TBT concentration in surviving fish was observed between the two strains. However, the intrinsic TBT concentration in dead KO-strain was significantly lower than that in WT-strain. These findings provide direct evidence, supporting the hypothesis that TBT-bp2 plays a critical role in the detoxification of TBT in fish.

Bioaccumulation and reproductive effects of fluorescent microplastics in medaka fish.

This study was performed to elucidate the uptake and bioaccumulation of polystyrene microplastics (PS-MPs) in Japanese medaka (freshwater fish) and Java medaka (marine fish), and to assess its impacts on the survival, reproduction, and gene expression of Japanese medaka. Both species were exposed to 2-µm fluorescent PS-MPs (107 beads/L) for 3 weeks. The bioaccumulation factor of PS-MPs for Java medaka was calculated at about 4 × 102, higher than that for Japanese medaka (about 1 × 102). The exposure had no significant effects on the survival and reproduction of Japanese medaka. The mRNA sequencing analysis showed that the expression of a few genes involved in the cell adhesion, xenobiotic metabolic process, brain development, and other functions in medaka intestines significantly changed after exposure. These results suggest that virgin PS-MPs can potentially accumulate in medaka intestines, but has limited toxicity to Japanese medaka at the concentration up to 107 beads/L.

Establishment of a Japanese medaka (Oryzias latipes) transgenic line expressing Takifugu rubripes pufferfish saxitoxin and tetrodotoxin binding protein 1, and evaluation of tributyltin toxicity via in ovo nanoinjection.

Pufferfish saxitoxin and tetrodotoxin binding proteins (PSTBPs) play an important role in the toxification of certain species of pufferfish. Recombinant Takifugu rubripes PSTBP1 (rTrub.PSTBP1) is reported to bind to tributyltin, and so it has been suggested that rTrub.PSTBP1 may reduce the toxicity of tributyltin. However, the role of PSTBP1 in vivo remains to be elucidated. Here, we established a transgenic medaka line showing whole-body Renilla reniformis green fluorescent protein and Trub.PSTBP1 expression, as confirmed by real-time polymerase chain reaction and mRNA-Seq analysis. mRNA-Seq analysis also showed that cytochrome P450 superfamily genes and the gene encoding ATP-binding cassette sub-family G member 2 were highly expressed in the transgenic medaka. Using embryos of the transgenic medaka line, we conducted an in ovo nanoinjection test to examine the effect of Trub.PSTBP1 in vivo, and obtained data suggesting that Trub.PSTBP1 expression may have reduced the toxicity of tributyltin in our transgenic medaka line. Our findings will be useful for future functional analyses of Trub.PSTBP1.