Language：English   Publisher：Antioxidants  

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.

DOI： 10.3390/antiox13070781

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Publisher：Limnology and Oceanography: Methods  

It is empirically known that fish exposed to harmful algal blooms (HABs) exhibit abnormal behavior. This might serve as a method for early detection of HABs. There has been no report of the detection of behavioral abnormalities of fish exposed to harmful algae using machine learning. In this study, the behavior of Oryzias javanicus (Java medaka) exposed in a stepwise manner to the HAB species Karenia mikimotoi at densities of 0 cells mL−1 (control), 1 × 103 cells mL−1 (nonlethal), and 5 × 103 cells mL−1 (sublethal) was recorded for 30 min at each cell density using two digital cameras connected to a software that tracked behavioral metrics of fish. The level of anomaly in the behavior of Java medaka was then analyzed using one-class support vector machines (OC-SVM) to determine whether the behavioral changes could be considered abnormal. The results revealed abnormal swimming behavior evidenced by an increase of swimming speed, a decrease of shoaling behavior, and a greater depth of swimming in Java medaka exposed especially to the sublethal K. mikimotoi density. The medaka exposed to K. mikimotoi also displayed physical deformities of their gills that were thought to have caused their abnormal behavior. This supposition was confirmed by further analysis using OC-SVM because the behavior of groups exposed to nonlethal and sublethal densities of K. mikimotoi were considered abnormal compared with that of the control groups. The results of this study show the possibility of using this system for early and real-time detection of HABs.

DOI： 10.1002/lom3.10613

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Language：English   Publisher：Wiley  

It is empirically known that fish exposed to harmful algal blooms (HABs) exhibit abnormal behavior. This might serve as a method for early detection of HABs. There has been no report of the detection of behavioral abnormalities of fish exposed to harmful algae using machine learning. In this study, the behavior of Oryzias javanicus (Java medaka) exposed in a stepwise manner to the HAB species Karenia mikimotoi at densities of 0 cells mL-<−1> (control), 1 × 10^3 cells mL^<−1> (nonlethal), and 5 × 10^3 cells mL^<−1> (sublethal) was recorded for 30 min at each cell density using two digital cameras connected to a software that tracked behavioral metrics of fish. The level of anomaly in the behavior of Java medaka was then analyzed using one-class support vector machines (OC-SVM) to determine whether the behavioral changes could be considered abnormal. The results revealed abnormal swimming behavior evidenced by an increase of swimming speed, a decrease of shoaling behavior, and a greater depth of swimming in Java medaka exposed especially to the sublethal K. mikimotoi density. The medaka exposed to K. mikimotoi also displayed physical deformities of their gills that were thought to have caused their abnormal behavior. This supposition was confirmed by further analysis using OC-SVM because the behavior of groups exposed to nonlethal and sublethal densities of K. mikimotoi were considered abnormal compared with that of the control groups. The results of this study show the possibility of using this system for early and real-time detection of HABs.

CiNii Research

Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Chemosphere  

There is global concern that microplastics may harm aquatic life. Here, we examined the effects of fine polystyrene microplastics (PS-MPs, 2-μm diameter, 0.1 mg/L, 2.5 × 107 particles/L) on the behavior and the microbiome (linked to brain–gut interaction) of a fish model using medaka, Oryzias latipes. We found that shoaling behavior was reduced in PS-MP-exposed medaka compared with control fish during the exposure period, but it recovered during a depuration period. There was no difference in swimming speed between the PS-MP-exposed and control groups during the exposure period. Analysis of the dominant bacterial population (those comprising ≥1% of the total bacterial population) in the gut of fish showed that exposure to PS-MPs tended to increase the relative abundance of the phylum Fusobacteria and the genus Vibrio. Furthermore, structural-equation modeling of gut bacteria on the basis of machine-learning data estimated strong relationship involved in the reduction of the functional bacterial species of minority (<1% of the total bacterial population) such as the genera Muribaculum (an undefined role), Aquaspirillum (a candidate for nitrate metabolism and magnetotactics), and Clostridium and Phascolarctobacterium (potential producers of short-chain fatty acids, influencing behavior by affecting levels of neurotransmitters) as a group of gut bacteria in association with PS-MP exposure. Our results suggest that fish exposure to fine microplastics may cause dysbiosis and ultimately cause social behavior disorders linked to brain–gut interactions. This effect could be connected to reduction of fish fitness in the ecosystem and reduced fish survival.

DOI： 10.1016/j.chemosphere.2024.141643

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Environmental Toxicology and Chemistry  

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.

DOI： 10.1002/etc.5713

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

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.

DOI： 10.1016/j.chemosphere.2023.139543

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Environmental Pollution  

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.

DOI： 10.1016/j.envpol.2023.122011

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Language：English   Publisher：Faculty of Agriculture, Kyushu University  

As one of the most commonly detected polluting drugs in the environment, diazepam (DZP) has been widely detected in rivers, soil, and organisms. To better understand the bioconcentration of DZP in fish and its impact on fish behaviors, we exposed both female and male medaka to a DZP (120 μg/L) for 7 days, and then transformed them to clean dechlorinated water for 3 days of depuration. Regardless of gender, DZP can rapidly accumulate in the brain and liver tissue and reach a stable phase within 24 hours of exposure. When the fish was transferred to purified water for purification, the concentration of DZP in the tissue was rapidly discharged, decreasing by one order of magnitude within 24 hours. The bioconcentration factors (BCF) of DZP in the brain and liver of males were calculated to be 10.47 and 19.58 L/kg, respectively, and those in females were calculated to be 6.12 and 6.03 L/kg. Compared with females, DZP accumulated more in the tissue of male fish, which exhibited a higher mortality rate during exposure. In addition, DZP can be significantly enriched in the ovaries of females, with a BCF of 10.54 L/kg. Furthermore, DZP exposure could significantly interfere with the interactive behaviors between males and females during the courtship, as indicated by the reduced duration of body contact and increased inter–individual distance. These findings highlight the need to study the transgenerational toxicity of DZP in Japanese medaka to assess its potential risk to fish populations accurately.

DOI： 10.5109/6796256

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Language：English   Publisher：Ecotoxicology  

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.

DOI： 10.1007/s10646-023-02700-4

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

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 17β-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 17β-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.

DOI： 10.1016/j.cbpc.2023.109660

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology  

The estuarine resident crustacean sand shrimp, Crangon uritai, has a higher tolerance to neonicotinoid insecticides than that of the kuruma prawns, Penaeus japonicus. However, the reason for the differential sensitivities between the two marine crustaceans remains to be understood. This study explored the mechanism underlying differential sensitivities based on insecticide body residues after exposing both said crustaceans to two insecticides (acetamiprid and clothianidin) with or without oxygenase inhibitor piperonyl butoxide (PBO) for 96 h. Two graded-concentration groups were formed; group H (1/15-1 times the 96-h LC50 values) and L (one-tenth the concentration of group H). Results showed that the internal concentration in survived specimens tended to be lower in sand shrimp than in kuruma prawns. Co-treatment of PBO with two neonicotinoids not only increased sand shrimp mortality in the H group, but also altered metabolism of acetamiprid into its metabolite, N-desmethyl acetamiprid. Furthermore, molting during the exposure period enhanced bioconcentration of insecticides, but not affects survival. Collectively, the higher tolerance of sand shrimp than that of kuruma prawns to the two neonicotinoids can be explained by lower bioconcentration potential and more involvement of oxygenase in their alleviating lethal toxicity.

DOI： 10.1016/j.cbpc.2023.109613

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

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.

DOI： 10.1016/j.chemosphere.2022.134403

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Aquatic Toxicology  

In aquatic arthropods, molting is a pivotal physiological process for normal development, but it may also expose them to higher risks from xenobiotics, because the organism may take up additional water during that time. This study aimed to assess the effects of molting on bioconcentration and survival after 96-h exposure to insecticide fipronil with or without oxygenase (CYP450s) inhibitor piperonyl butoxide (PBO) of two estuarine resident marine crustacean species: the sand shrimp Crangon uritai and the kuruma prawn Penaeus japonicus, with 96-h LC50 value of fipronil = 2.0 µg/L and 0.2 µg/L, respectively. Two graded concentrations included group high (H) (equivalent to the 96-h LC50 values) and low (L) (one-tenth of the H group concentration). Molting and survival were individually checked, and internal concentrations of fipronil and its metabolites (fipronil desulfinyl, fipronil sulfide, fipronil sulfone) were measured. The results showed that, only fipronil and fipronil sulfone were detected from organism, and that internal concentrations of these insecticides in molted specimens were higher than those of unmolted ones but comparable with those of dead ones. Accordingly, mortality was more frequent in molted specimens than those that were unmolted. Furthermore, involvement of oxygenase and higher lethal body burden threshold may confer higher tolerance to fipronil in sand shrimp than in the kuruma prawn. This study is the first to demonstrate that the body-residue-based approach is useful for deciphering the causal factors underlying fipronil toxicity, but highlights the need to consider physiological factors in arthropods, which influence and lie beyond body burden, molting and drug metabolism.

DOI： 10.1016/j.aquatox.2022.106172

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Aquatic Toxicology  

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.

DOI： 10.1016/j.aquatox.2022.106129

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.aquatox.2021.105907

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ecoenv.2021.112007

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.aquatox.2020.105643

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DOI： 10.1016/j.marpolbul.2020.111601

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DOI： 10.1016/j.marpolbul.2020.111445

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DOI： 10.1080/00318884.2020.1816041

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DOI： 10.1016/j.cbpc.2020.108785

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Event date： 2024.9

Presentation type：Oral presentation (general)  

Venue：Tianjin   Country：China  

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Event date： 2024.9

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Venue：東京   Country：Japan  

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Effect of Anthracene Spiked Polystyrene Microplastics on Anthracene Accumulation in Java Medaka

Event date： 2022.9

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Event date： 2015.9

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