Assessment of environmental exposure to betamethasone on the reproductive function of female Japanese medaka (Oryzias latipes).

Betamethasone has been extensively used in medicine in recent years and poses potential hazards to aquatic organisms. This study investigated the reproductive toxic effects of betamethasone exposure in fish, employing female Japanese medaka (Oryzias latipes) as a model. Betamethasone exposure at environmentally relevant concentrations (0, 20, 200, and 2000 ng/L) for a period of 15 weeks resulted in its high accumulation in the ovary, leading to abnormal oogenesis in female Japanese medaka. The production of gonadotropins (LH and FSH) in the pituitary gland was inhibited, and sex steroid biosynthesis in the ovary was significantly influenced at the transcriptional level. The imbalance of androgens and estrogens resulted in a decrease in the E2/T ratio and hepatic VTG synthesis, and the suppression of estrogen receptor signaling was also induced. Furthermore, betamethasone exposure delayed spawning and reduced fertility in the F0 generation, and had detrimental effects on the fertilization rate and hatchability of the F1 generation. Our results showed that environmental betamethasone had the potential to adversely affect female fertility and steroid hormone dynamics in fish.

Neuropeptide Y receptor Y8b (npy8br) regulates feeding and digestion in Japanese medaka (Oryzias latipes) larvae: evidence from gene knockout. / 神经肽受体Y8b（npy8br）调节日本青鳉（Oryzias latipes）仔鱼的摄食和消化： æ¥è‡ªåŸºå› æ•²é™¤çš„è¯æ®.

Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor with two subtypes, NPY8AR and NPY8BR. Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation; this has been found in only a few fish, at present. In order to better understand the physiological function of npy8br, especially in digestion, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate npy8br-/- Japanese medaka (Oryzias latipes). We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability, ultimately affecting their growth. Specifically, npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes (npy and agrp). npy8br-/- medaka larvae fed for 10 d (10th day of feeding) still had incompletely digested brine shrimp (Artemia nauplii) in the digestive tract 8 h after feeding, the messenger RNA (mRNA) expression levels of digestion-related genes (amy, lpl, ctra, and ctrb) were significantly decreased, and the activity of amylase, trypsin, and lipase also significantly decreased. The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes (gh and igf1). Hematoxylin and eosin (H&E) sections of intestinal tissue showed that npy8br-/- medaka larvae had damaged intestine, thinned intestinal wall, and shortened intestinal villi. So far, this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.

Characterization of medaka (Oryzias latipes) AHRs and the comparison of two model fishes-Medaka vs. zebrafish: The subform-specific sensitivity to dioxin.

Dioxins and the emerging dioxin-like compounds (DLCs) have recruited increasing concerns about their environmental contamination, toxicity, health impacts, and mechanisms. Based on the structural similarity of dioxins and many DLCs, their toxicity was predominantly mediated by the dioxin receptor (aryl hydrocarbon receptor, AHR) in animals (including human), which can be different in expression and function among species and then possibly produce the species-specific risk or toxicity. To date, characterizing the AHR of additional species other than human and rodents can increase the accuracy of toxicity/risk evaluation and increase knowledge about AHR biology. As a key model, the medaka AHR has not been clearly characterized. Through genome survey and phylogenetic analysis, we identified four AHRs (olaAHR1a, olaAHR1b, olaAHR2a, and olaAHR2b) and two ARNTs (olaARNT1 and olaARNT2). The medaka AHR pathway was conserved in expression in nine tested tissues, of which olaAHR2a represented the predominant subform with greater abundance. Medaka AHRs and ARNTs were functional and could be efficiently transactivated by the classical dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), although olaAHR1a did not seem to cooperate with olaARNT2. In terms of function/sensitivity, the EC50 values of medaka olaAHR1a (9.01 ± 1.43 nM), olaAHR1b (4.00 ± 1.10 nM), olaAHR2a (8.75 ± 3.34 nM), and olaAHR2b (3.06 ± 0.81 nM) showed slight differences; however, they were all at the nM level. The sensitivity of four medaka AHRs to TCDD was similar to that of zebrafish dreAHR2 (the dominant form, EC50 = 3.14 ± 4.19 nM), but these medaka AHRs were more sensitive than zebrafish dreAHR1b (EC50 = 27.05 ± 18.51 nM). The additional comparison also indicated that the EC50 values in various species were usually within the nM range, but AHRs of certain subforms/species can vary by one or two orders of magnitude. In summary, the present study will enhance the understanding of AHR and help improve research on the ecotoxicity of dioxins/DLCs.

Characterizing medaka visual features using a high-throughput optomotor response assay.

Medaka fish (Oryzias latipes) is a powerful model to study genetics underlying the developmental and functional traits of the vertebrate visual system. We established a simple and high-throughput optomotor response (OMR) assay utilizing medaka larvae to study visual functions including visual acuity and contrast sensitivity. Our assay presents multiple adjustable stripes in motion to individual fish in a linear arena. For that the OMR assay employs a tablet display and the Fish Stripes software to adjust speed, width, color, and contrast of the stripes. Our results demonstrated that optomotor responses were robustly induced by black and white stripes presented from below in the linear-pool-arena. We detected robust strain specific differences in the OMR when comparing long established medaka inbred strains. We observed an interesting training effect upon the initial exposure of larvae to thick stripes, which allowed them to better respond to narrower stripes. The OMR setup and protocol presented here provide an efficient tool for quantitative phenotype mapping, addressing visual acuity, trainability of cortical neurons, color sensitivity, locomotor response, retinal regeneration and others. Our open-source setup presented here provides a crucial prerequisite for ultimately addressing the genetic basis of those processes.

The Hox code responsible for the patterning of the anterior vertebrae in zebrafish.

The vertebral column is a characteristic structure of vertebrates. Genetic studies in mice have shown that Hox-mediated patterning plays a key role in specifying discrete anatomical regions of the vertebral column. Expression pattern analyses in several vertebrate embryos have provided correlative evidence that the anterior boundaries of Hox expression coincide with distinct anatomical vertebrae. However, because functional analyses have been limited to mice, it remains unclear which Hox genes actually function in vertebral patterning in other vertebrates. In this study, various zebrafish Hox mutants were generated for loss-of-function phenotypic analysis to functionally decipher the Hox code responsible for the zebrafish anterior vertebrae between the occipital and thoracic vertebrae. We found that Hox genes in HoxB- and HoxC-related clusters participate in regulating the morphology of the zebrafish anterior vertebrae. In addition, medaka hoxc6a was found to be responsible for anterior vertebral identity, as in zebrafish. Based on phenotypic similarities with Hoxc6 knockout mice, our results suggest that the Hox patterning system, including at least Hoxc6, may have been functionally established in the vertebral patterning of the common ancestor of ray-finned and lobe-finned fishes.

Gsdf is not indispensable for male differentiation in the medaka species Oryzias hubbsi.

Sex determination mechanisms differ widely among vertebrates, particularly in fish species, where diverse sex chromosomes and sex-determining genes have evolved. However, the sex-differentiation pathways activated by these sex-determining genes appear to be conserved. Gonadal soma-derived growth factor (Gsdf) is one of the genes conserved across teleost fish, especially in medaka fishes of the genus Oryzias, and is implicated in testis differentiation and germ cell proliferation. However, its role in sex differentiation remains unclear. In this study, we investigated Gsdf function in Oryzias hubbsi, a species with a ZW sex-determination system. We confirmed its male-dominant expression, as in other species. However, histological analyses revealed no male-to-female sex reversal in Gsdf-knockout fish, contrary to findings in other medaka species. Genetic sex determination remained intact without Gsdf function, indicating a Gsdf-independent sex-differentiation pathway in O. hubbsi. Instead, Gsdf loss led to germ cell overproliferation in both sexes and accelerated onset of meiosis in testes, suggesting a role in germ cell proliferation. Notably, the feminizing effect of germ cells observed in O. latipes was absent, suggesting diverse germ cell-somatic cell relationships in Oryzias gonad development. Our study highlights species-specific variations in the molecular pathways governing sex determination and differentiation, emphasizing the need for further exploration to elucidate the complexities of sexual development.

Sperm handling and management in the teleost model fish Japanese medaka (Oryzias latipes).

Japanese medaka (Oryzias latipes) has been used as a model organism in different research fields, including reproductive physiology. Sperm motility is the most important marker for male fertility in fish and, thus, reproduction success. However, because of small volume of ejaculate and short motility duration, it is still challenging to manage the sperm collection and analysis in small model fish. In the present study, we aimed to investigate sperm motility and to optimize sperm collection, short-term sperm storage, and cryopreservation in Japanese medaka (Oryzias latipes). Using two different approaches for sperm collection: testes dissection and abdominal massage, different housing conditions and activating the sperm with different activation solutions, we investigated immediate sperm motility. In the second part of this study, we used different osmolalities of immobilization solution, Hank's Balanced Salt Solution (HBSS) for sperm storage at 0, 2 and 3 h after sperm collection. Finally, the sperm were cryopreserved using methanol as cryoprotectant and HBSS as extender at two different osmolalities, and post-thaw sperm motility was investigated. The highest post-activating sperm motility was achieved in the groups activated by the extender at 300 mOsm/kg. The quality of sperm remained unaffected by co-housing with females or with males only. Furthermore, Hanks' Balanced Salt Solution (HBSS) with an osmolality of 600 mOsm/kg demonstrated its efficacy as a suitable extender for sperm storage, preserving motility and progressivity for 3 h. The highest post-thaw motility was around 35%. There were no significant differences between post-thaw motility in different groups. We also found that post-thaw incubation on ice can maintain the motility of the sperm for up to one hour after thawing.

Multigenerational exposure to temperature influences mitochondrial oxygen fluxes in the Medaka fish (Oryzias latipes).

AIM: Thermal sensitivity of cellular metabolism is crucial for animal physiology and survival under climate change. Despite recent efforts, effects of multigenerational exposure to temperature on the metabolic functioning remain poorly understood. We aimed at determining whether multigenerational exposure to temperature modulate the mitochondrial respiratory response of Medaka fish. METHODS: We conducted a multigenerational exposure with Medaka fish reared multiple generations at 20 and 30°C (COLD and WARM fish, respectively). We then measured the oxygen consumption of tail muscle at two assay temperatures (20 and 30°C). Mitochondrial function was determined as the respiration supporting ATP synthesis (OXPHOS) and the respiration required to offset proton leak (LEAK(Omy)) in a full factorial design (COLD-20°C; COLD-30°C; WARM-20°C; WARM-30°C). RESULTS: We found that higher OXPHOS and LEAK fluxes at 30°C compared to 20°C assay temperature. At each assay temperature, WARM fish had lower tissue oxygen fluxes than COLD fish. Interestingly, we did not find significant differences in respiratory flux when mitochondria were assessed at the rearing temperature of the fish (i.e., COLD-20°C vs. WARM -30°C). CONCLUSION: The lower OXPHOS and LEAK capacities in warm fish are likely the result of the multigenerational exposure to warm temperature. This is consistent with a modulatory response of mitochondrial capacity to compensate for potential detrimental effects of warming on metabolism. Finally, the absence of significant differences in respiratory fluxes between COLD-20°C and WARM-30°C fish likely reflects an optimal respiration flux when organisms adapt to their thermal conditions.

Long-term carbaryl exposure leads to behavioral abnormalities and reproductive toxicity in male marine medaka through apoptosis-mediated HPA and HPG axes dysregulation.

Carbaryl is a widely used carbamate pesticide that has been detected in the marine environment, but its effects on marine fish are still unknown. This study was aimed to investigate the effects of long-term exposure of carbaryl on male marine medaka. For this purpose, we set up five exposure concentration groups of 0, 0.1, 1, 10, and 100 µg/L for 180 days. On the one hand, we observed increased aggression and decreased ability to avoid predators in males after exposure, which was affected by the levels of HPA-axis hormones, especially decreased cortisol level. On the other hand, after exposure, HPG axis hormone levels and gene transcription levels were disturbed. Males exhibited a decreased gonadosomatic index and a notable reduction in mature sperm proportion and the F1 generation displayed a significant increase in malformation rate. Additionally, the number of apoptotic cells and the transcription level of apoptosis-related genes in the brains of male marine medaka substantially increased after exposure. Apoptosis of brain cells may be responsible for the disturbance of HPA and HPG axes, consequently leading to behavioral and reproductive abnormalities. These findings provide novel insights into evaluating the toxic effects of carbaryl on male marine medaka and emphasizing the criticality of exploring the potential environmental risks posed by carbaryl in the marine environment, thus providing toxicity value basis for further strengthening marine environmental monitoring and the protection of biological resources.

Comparative effects of different metals on the Japanese medaka embryos and larvae.

Rapid evaluation of the toxicity of metals using fish embryo acute toxicity is facilitative to ecological risk assessment of aquatic organisms. However, this approach has seldom been utilized for the comparative study on the effects of different metals to fish. In this study, acute and sub-chronic tests were used to compare the toxicity of Se(IV) and Cd in the embryos and larvae of Japanese medaka (Oryzias latipes). The embryos with different levels of dechorionation and/or pre-exposure were also exposed to Se(IV) and Cd at various concentrations. The results showed that the LC50-144 h of Cd was 1.3-5.2 folds higher than that of Se(IV) for the embryos. In contrast, LC50-96 h of Se(IV) were 200-400 folds higher than that of Cd for the larvae. Meanwhile, dechorionated embryos were more sensitive to both Se and Cd than the intact embryos. At elevated concentrations, both Se and Cd caused mortality and deformity in the embryos and larvae. In addition, pre-exposure to Cd at the embryonic stages enhanced the resistance to Cd in the larvae. However, pre-exposure to Se(IV) at the embryonic stages did not affect the toxicity of Se(IV) to the larvae. This study has distinguished the nuance differences in effects between Se(IV) and Cd after acute and sub-chronic exposures with/without chorion. The approach might have a potential in the comparative toxicology of metals (or other pollutants) and in the assessment of their risks to aquatic ecosystems.

Teleost Hox code defines regional identities competent for the formation of dorsal and anal fins.

The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position.

Identification of the FSH-RH as the other gonadotropin-releasing hormone.

In vertebrates, folliculogenesis and ovulation are regulated by two distinct pituitary gonadotropins: follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Currently, there is an intriguing consensus that a single hypothalamic neurohormone, gonadotropin-releasing hormone (GnRH), regulates the secretion of both FSH and LH, although the required timing and functions of FSH and LH are different. However, recent studies in many non-mammalian vertebrates indicated that GnRH is dispensable for FSH function. Here, by using medaka as a model teleost, we successfully identify cholecystokinin as the other gonadotropin regulator, FSH-releasing hormone (FSH-RH). Our histological and in vitro analyses demonstrate that hypothalamic cholecystokinin-expressing neurons directly affect FSH cells through the cholecystokinin receptor, Cck2rb, thereby increasing the expression and release of FSH. Remarkably, the knockout of this pathway minimizes FSH expression and results in a failure of folliculogenesis. Here, we propose the existence of the "dual GnRH model" in vertebrates that utilize both FSH-RH and LH-RH.

Biomass-derived carbon dots as fluorescent quantum probes to visualize and modulate inflammation.

Quantum dots, which won the Nobel Prize in Chemistry, have recently gained significant attention in precision medicine due to their unique properties, such as size-tunable emission, high photostability, efficient light absorption, and vibrant luminescence. Consequently, there is a growing demand to identify new types of quantum dots from various sources and explore their potential applications as stimuli-responsive biosensors, biomolecular imaging probes, and targeted drug delivery agents. Biomass-waste-derived carbon quantum dots (CQDs) are an attractive alternative to conventional QDs, which often require expensive and toxic precursors, as they offer several merits in eco-friendly synthesis, preparation from renewable sources, and cost-effective production. In this study, we evaluated three CQDs derived from biomass waste for their potential application as non-toxic bioimaging agents in various cell lines, including human dermal fibroblasts, HeLa, cardiomyocytes, induced pluripotent stem cells, and an in-vivo medaka fish (Oryzias latipes) model. Confocal microscopic studies revealed that CQDs could assist in visualizing inflammatory processes in the cells, as they were taken up more by cells treated with tumor necrosis factor-α than untreated cells. In addition, our quantitative real-time PCR gene expression analysis has revealed that citric acid-based CQDs can potentially reduce inflammatory markers such as Interleukin-6. Our studies suggest that CQDs have potential as theragnostic agents, which can simultaneously identify and modulate inflammatory markers and may lead to targeted therapy for immune system-associated diseases.

Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes.

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

Japanese medaka (Oryzias latipes) Nectin4 plays an important role against red spotted grouper nervous necrosis virus infection.

Nectins are adhesion molecules that play a crucial role in the organization of epithelial and endothelial junctions and function as receptors for the entry of herpes simplex virus. However, the role of Nectin4 remains poorly understood in fish. In this study, nectin4 gene was cloned from medaka (OlNectin4). OlNectin4 was located on chromosome 18 and contained 11 exons, with a total genome length of 25754 bp, coding sequences of 1689 bp, coding 562 amino acids and a molecular weight of 65.5 kDa. OlNectin4 contained four regions, including an Immunoglobulin region, an Immunoglobulin C-2 Type region, a Transmembrane region and a Coiled coil region. OlNectin4 shared 47.18 % and 25.00 % identity to Paralichthys olivaceus and Mus musculus, respectively. In adult medaka, the transcript of nectin4 was predominantly detected in gill. During red spotted grouper nervous necrosis virus (RGNNV) infection, overexpression of OlNectin4 in GE cells significantly increased viral gene transcriptions. Meanwhile, Two mutants named OlNectin4△4 (+4 bp) and OlNectin4△7 (-7 bp) medaka were established using CRISPR-Cas9 system. Nectin4-KO medaka had higher mortality than WT after infected with RGNNV. Moreover, the expression of RGNNV RNA2 gene in different tissues of the Nectin4-KO were higher than WT medaka after challenged with RGNNV. The brain and eye of Nectin4-KO medaka which RGNNV mainly enriched, exhibited significantly higher expression of interferon signaling genes than in WT. Taken together, the OlNectin4 plays a complex role against RGNNV infection by inducing interferon responses for viral clearance.

Medaka Terb1 Mutant Displays Defects of Synaptonemal Complex Formation and Sexual Difference in Gametogenesis.

Formation of the synaptonemal complex (SC) is a prerequisite for proper recombination and chromosomal segregation during meiotic prophase I. One mechanism that ensures SC formation is chromosomal movement, which is driven by the force derived from cytoskeletal motors. Here, we report the phenotype of medaka mutants lacking the telomere repeat binding bouquet formation protein 1 (TERB1), which, in combination with the SUN/KASH protein, mediates chromosomal movement by connecting telomeres and cytoskeletal motors. Mutations in the terb1 gene exhibit defects in SC formation in medaka. Although SC formation was initiated, as seen by the punctate lateral elements and fragmented transverse filaments, it was not completed in the terb1 mutant meiocytes. The mutant phenotype further revealed that the introduction of double strand breaks was independent of synapsis completion. In association with these phenotypes, meiocytes in both the ovaries and testes exhibited an aberrant arrangement of homologous chromosomes. Interestingly, although oogenesis halted at the zygotene-like stage in terb1 mutant, testes continued to produce sperm-like cells with aberrant DNA content. This indicates that the mechanism of meiotic checkpoint is sexually different in medaka, similar to the mammalian checkpoint in which oogenesis proceeds while spermatogenesis is arrested. Moreover, our results suggest that spermatogenesis is mechanistically dissociable from meiosis.

Toxicity and speciation of inorganic arsenics and their adverse effects on in vivo endpoints and oxidative stress in the marine medaka Oryzias melastigma.

Here, we investigate the effects of acute and chronic exposure to arsenate (AsV) and arsenite (AsIII) in the marine medaka Oryzias melastigma. In vivo effects, biotransformation, and oxidative stress were studied in marine medaka exposed to the two inorganic arsenics for 4 or 28 days. An investigation of embryonic development revealed no effect on in vivo parameters, but the hatching rate increased in the group exposed to AsIII. Exposure to AsIII also caused the greatest accumulation of arsenic in medaka. For acute exposure, the ratio of AsV to AsIII was higher than that of chronic exposure, indicating that bioaccumulation of inorganic arsenic can induce oxidative stress. The largest increase in oxidative stress was observed following acute exposure to AsIII, but no significant degree of oxidative stress was induced by chronic exposure. During acute exposure to AsV, the increase in the enzymatic activity of glutathione-S-transferase (GST) was twice as high compared with exposure to AsIII, suggesting that GST plays an important role in the initial detoxification process. In addition, an RNA-seq-based ingenuity pathway analysis revealed that acute exposure to AsIII may be related to cell-cycle progression. A network analysis using differentially expressed genes also revealed a potential link between the generation of inflammatory cytokines and oxidative stress due to arsenic exposure.

New insights into the impact of leachates from in-field collected plastics on aquatic invertebrates and vertebrates.

The impact of leachates from micronized beached plastics of the Mediterranean Sea and Atlantic Ocean on coastal marine ecosystems was investigated by using a multidisciplinary approach. Chemical analysis and ecotoxicological tests on phylogenetically distant species were performed on leachates from the following plastic categories: bottles, pellets, hard plastic (HP) containers, fishing nets (FN) and rapido trawling rubber (RTR). The bacteria Alivibrio fischeri, the nauplii of the crustaceans Amphibalanus amphitrite and Acartia tonsa, the rotifer Brachionus plicatilis, the embryos of the sea urchin Paracentrotus lividus, the ephyrae of the jellyfish Aurelia sp. and the larvae of the medaka Oryzias latipes were exposed to different concentrations of leachates to evaluate lethal and sub-lethal effects. Thirty-one additives were identified in the plastic leachates; benzophenone, benzyl butyl phthalate and ethylparaben were present in all leachates. Ecotoxicity of leachates varied among plastic categories and areas, being RTR, HP and FN more toxic than plastic bottles and pellets to several marine invertebrates. The ecotoxicological results based on 13 endpoints were elaborated within a quantitative weight of evidence (WOE) model, providing a synthetic hazard index for each data typology, before their integrations in an environmental risk index. The WOE assigned a moderate and slight hazard to organisms exposed to leachates of FN and HP collected in the Mediterranean Sea respectively, and a moderate hazard to leachates of HP from the Atlantic Ocean. No hazard was found for pellet, bottles and RTR. These findings suggest that an integrated approach based on WOE on a large set of bioassays is recommended to get a more reliable assessment of the ecotoxicity of beached-plastic leachates. In addition, the additives leached from FN and HP should be further investigated to reduce high concentrations and additive types that could impact marine ecosystem health.

Behavioral profile alterations and predation susceptibility of Japanese medaka fish exposed to phenytoin, an antiepileptic drug.

Antiepileptic drugs, such as phenytoin, are often leaked into aquatic systems through sewage facilities due to their low metabolic rate. Fish, such as the Japanese medaka (Oryzias latipes), demonstrate abnormal swimming behavior such as equilibrium abnormalities, rotational behavior, and vertical swimming, when exposed to phenytoin. Therefore, it is hypothesized that predator avoidance may be hindered. This study aimed to investigate the effects of phenytoin exposure-induced behavioral abnormalities in predator avoidance in Japanese medaka. The results showed that individuals with behavioral abnormalities had a reduced ability to avoid danger. Furthermore, the fish demonstrated a delayed recognition reaction to approaching predators. Additionally, predatory fish, such as silver pike characin (Ctenolucius hujeta), were more likely to prey upon abnormal individuals. In conclusion, the fish exposed to phenytoin demonstrated behavioral changes that increased its predation risk. This study is the first to determine the effects of behavioral abnormalities in Japanese medaka which was induced after phenytoin exposure on predator risk avoidance.

The decision of male medaka to mate or fight depends on two complementary androgen signaling pathways.

Adult male animals typically court and attempt to mate with females, while attacking other males. Emerging evidence from mice indicates that neurons expressing the estrogen receptor ESR1 in behaviorally relevant brain regions play a central role in mediating these mutually exclusive behavioral responses to conspecifics. However, the findings in mice are unlikely to apply to vertebrates in general because, in many species other than rodents and some birds, androgens-rather than estrogens-have been implicated in male behaviors. Here, we report that male medaka (Oryzias latipes) lacking one of the two androgen receptor subtypes (Ara) are less aggressive toward other males and instead actively court them, while those lacking the other subtype (Arb) are less motivated to mate with females and conversely attack them. These findings indicate that, in male medaka, the Ara- and Arb-mediated androgen signaling pathways facilitate appropriate behavioral responses, while simultaneously suppressing inappropriate responses, to males and females, respectively. Notably, males lacking either receptor retain the ability to discriminate the sex of conspecifics, suggesting a defect in the subsequent decision-making process to mate or fight. We further show that Ara and Arb are expressed in intermingled but largely distinct populations of neurons, and stimulate the expression of different behaviorally relevant genes including galanin and vasotocin, respectively. Collectively, our results demonstrate that male teleosts make adaptive decisions to mate or fight as a result of the activation of one of two complementary androgen signaling pathways, depending on the sex of the conspecific that they encounter.