Comprehensive effects of salinity, dissolved organic carbon and copper on mortality, osmotic regulation and bioaccumulation of copper in Oryzias melastigma.

Cu, as an essential and toxic element, has gained widespread attention. Both salinity and dissolved organic carbon (DOC) are known to influence Cu toxicity in marine organisms. However, the intricate interplay between these factors and their specific influence on Cu toxicity remains ambiguous. So, this study conducted toxicity tests of Cu on Oryzias melastigma. The experiments involved three salinity levels (10, 20, and 30 ppt) and three DOC levels (0, 1, and 5 mg/L) to comprehensively investigate the underlying mechanisms of toxicity. The complex toxic effects were analyzed by mortality, NKA activity, net Na+ flux and Cu bioaccumulation in O. melastigma. The results indicate that Cu toxicity is notably influenced by both DOC and salinity. Interestingly, the discernible variation in Cu toxicity across different DOC levels diminishes as salinity levels increase. The presence of DOC enhances the impact of salinity on Cu toxicity, especially at higher Cu concentrations. Additionally, Visual MINTEQ was utilized to elucidate the chemical composition of Cu, revealing that DOC had a significant impact on Cu forms. Furthermore, we observed that fluctuations in salinity lead to the inhibition of Na+/K+-ATPase (NKA) activity, subsequently hindering the inflow of Na+. The effects of salinity and DOC on the bioaccumulation of copper were not significant. The influence of salinity on Cu toxicity is mainly through its effect on the osmotic regulation and biophysiology of O. melastigma. Additionally, DOC plays a crucial role in the different forms of Cu. Moreover, DOC-Cu complexes can be utilized by organisms. This study contributes to understanding the mechanism of copper's biological toxicity in intricate marine environments and serves as a valuable reference for developing marine water quality criteria for Cu.

Interindividual behavioural variation in response to elevated CO2 predicts mRNA transcript abundance of genes related to acid-base regulation in medaka (Oryzias latipes).

Rising carbon dioxide (CO2) in aquatic ecosystems due to climate change is a challenge for aquatic ectotherms. We examined whether interindividual variation in behavioural responses to CO2 could predict how a teleost fish would respond to elevated CO2 for multiple phenotypic and molecular traits. To this end, we first quantified behavioural responses of individuals exposed to acute elevated CO2, and used these to assign individuals as either high or low responders relative to the population mean. Subsequently, we exposed both high and low responders to elevated CO2 for 6 weeks and quantified the effect on body condition, behaviour, and mRNA transcript responses of gill and liver genes associated with relevant physiological processes. Generally, we found few relationships between the phenotypic groups and body condition and behaviour following the CO2 exposure period; however, stark differences between the phenotypic groups with respect to gene transcripts from each tissue related to various processes were found, mostly independent of CO2 exposure. The most pronounced changes were in the gill transcripts related to acid-base regulation, suggesting that the observed behavioural variation used to assign fish to phenotypic groups may have an underlying molecular origin. Should the link between behaviour and gene transcripts be shown to have a fitness advantage and be maintained across generations, interindividual variation in behavioural responses to acute CO2 exposure may be a viable and non-invasive tool to predict future population responses to elevated aquatic CO2.

A high-quality chromosome-level genome assembly of the Chinese medaka Oryzias sinensis.

Oryzias sinensis, also known as Chinese medaka or Chinese ricefish, is a commonly used animal model for aquatic environmental assessment in the wild as well as gene function validation or toxicology research in the lab. Here, a high-quality chromosome-level genome assembly of O. sinensis was generated using single-tube long fragment read (stLFR) reads, Nanopore long-reads, and Hi-C sequencing data. The genome is 796.58 Mb, and a total of 712.17 Mb of the assembled sequences were anchored to 23 pseudo-chromosomes. A final set of 22,461 genes were annotated, with 98.67% being functionally annotated. The Benchmarking Universal Single-Copy Orthologs (BUSCO) benchmark of genome assembly and gene annotation reached 95.1% (93.3% single-copy) and 94.6% (91.7% single-copy), respectively. Furthermore, we also use ATAC-seq to uncover chromosome transposase-accessibility as well as related genome area function enrichment for Oryzias sinensis. This study offers a new improved foundation for future genomics research in Chinese medaka.

Sexually dimorphic dynamics of the microtubule network in medaka (Oryzias latipes) germ cells.

Gametogenesis is the process through which germ cells differentiate into sexually dimorphic gametes, eggs and sperm. In the teleost fish medaka (Oryzias latipes), a germ cell-intrinsic sex determinant, foxl3, triggers germline feminization by activating two genetic pathways that regulate folliculogenesis and meiosis. Here, we identified a pathway involving a dome-shaped microtubule structure that may be the basis of oocyte polarity. This structure was first established in primordial germ cells in both sexes, but was maintained only during oogenesis and was destabilized in differentiating spermatogonia under the influence of Sertoli cells expressing dmrt1. Although foxl3 was dispensable for this pathway, dazl was involved in the persistence of the microtubule dome at the time of gonocyte development. In addition, disruption of the microtubule dome caused dispersal of bucky ball RNA, suggesting the structure may be prerequisite for the Balbiani body. Collectively, the present findings provide mechanistic insight into the establishment of sex-specific polarity through the formation of a microtubule structure in germ cells, as well as clarifying the genetic pathways implementing oocyte-specific characteristics.

Detrimental impacts and QSAR baseline toxicity assessment of Japanese medaka embryos exposed to methylparaben and its halogenated byproducts.

Despite the theoretical risk of forming halogenated methylparabens (halo-MePs) during water chlorination in the absence or presence of bromide ions, there remains a lack of in vivo toxicological assessments on vertebrate organisms for halo-MePs. This research addresses these gaps by investigating the lethal (assessed by embryo coagulation) or sub-lethal (assessed by hatching success/heartbeat rate) toxicity and teratogenicity (assessed by deformity rate) of MeP and its mono- and di-halogen derivatives (Cl- or Br-) using Japanese medaka embryos. In assessing selected apical endpoints to discern patterns in physiological or biochemical alterations, heightened toxic impacts were observed for halo-MePs compared to MeP. These include a higher incidence of embryo coagulation (4-36 fold), heartbeat rate decrement (11-36 fold), deformity rate increment (32-223 fold), hatching success decrement (11-59 fold), and an increase in Reactive Oxygen Species (ROS) level (1.2-7.4 fold)/Catalase (CAT) activity (1.7-2.8 fold). Experimentally determined LC50 values are correlated and predicted using a Quantitative Structure Activity Relationship (QSAR) based on the speciation-corrected liposome-water distribution ratio (Dlipw, pH 7.5). The QSAR baseline toxicity aligns well with (sub)lethal toxicity and teratogenicity, as evidenced by toxic ratio (TR) analysis showing TR < 10 for MeP exposure in all cases, while significant specific or reactive toxicity was found for halo-MeP exposure, with TR > 10 observed (excepting three values). Our extensive findings contribute novel insights into the intricate interplay of embryonic toxicity during the early-life-stage of Japanese medaka, with a specific focus on highlighting the potential hazards associated with halo-MePs compared to the parent compound MeP.

Toxic effects of environmental concentration Bisphenol AF exposure on the survival, growth and reproduction of adult male Oryzias curvinotus.

Bisphenol AF (BPAF) is a novel environmental endocrine disruptor, and is widely detected in the aquatic environment, which is a potential threat to the health of fish. In this study, male Oryzias curvinotus were exposed to environmental concentrations (0.93 and 9.33 µg/L) of BPAF for 21 days. The effects of BPAF on survival, growth, reproduction, liver and testis histology, and gene transcriptional profiles of O. curvinotus were investigated. The results showed that the survival rate of male O. curvinotus slight decrease with increasing BPAF concentration, and there was no significant effect on body length, body weight, and K-factor. BPAF (9.33 µg/L) caused significant changes in testicular structure and reduced spermatid count in O. curvinotus. Changes in transcript levels of some antioxidant-related genes in gills and liver following BPAF exposure, imply an effect of BPAF on the immune system. After BPAF exposure, chgs and vtgs were up-regulated, validating the estrogenic effect of BPAF. In the hypothalamic - pituitary - gonadal axis (HPG) results, erα, erγ and cyp19a1b were all up-regulated in the brain, and the 0.93 µg/L BPAF group was more up-regulated than the 9.33 µg/L BPAF group. In testis, BPAF significantly up-regulated the mRNA expression level of cyp17a1 and cyp11b, while significantly down-regulated mRNA expression level of cyp11a, and cyp19a1 was significantly down-regulated only in the 0.93 µg/L BPAF group. In conclusion, environmental levels of BPAF have adverse effects on the survival and reproduction of O. curvinotus, and the potential toxic effects of environmental levels of BPAF cannot be ignored.

Alteration of shoaling behavior and dysbiosis in the gut of medaka (Oryzias latipes) exposed to 2-µm polystyrene microplastics.

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.

Chemical range recognized by the ligand-binding domain in a representative amino acid-sensing taste receptor, T1r2a/T1r3, from medaka fish.

Taste receptor type 1 (T1r) proteins are responsible for recognizing nutrient chemicals in foods. In humans, T1r2/T1r3 and T1r1/T1r3 heterodimers serve as the sweet and umami receptors that recognize sugars or amino acids and nucleotides, respectively. T1rs are conserved among vertebrates, and T1r2a/T1r3 from medaka fish is currently the only member for which the structure of the ligand-binding domain (LBD) has been solved. T1r2a/T1r3 is an amino acid receptor that recognizes various l-amino acids in its LBD as observed with other T1rs exhibiting broad substrate specificities. Nevertheless, the range of chemicals that are recognized by T1r2a/T1r3LBD has not been extensively explored. In the present study, the binding of various chemicals to medaka T1r2a/T1r3LBD was analyzed. A binding assay for amino acid derivatives verified the specificity of this protein to l-α-amino acids and the importance of α-amino and carboxy groups for receptor recognition. The results further indicated the significance of the α-hydrogen for recognition as replacing it with a methyl group resulted in a substantially decreased affinity. The binding ability to the protein was not limited to proteinogenic amino acids, but also to non-proteinogenic amino acids, such as metabolic intermediates. Besides l-α-amino acids, no other chemicals showed significant binding to the protein. These results indicate that all of the common structural groups of α-amino acids and their geometry in the l-configuration are recognized by the protein, whereas a wide variety of α-substituents can be accommodated in the ligand binding sites of the LBDs.

Evidence for hybridization-driven heteroplasmy maintained across generations in a ricefish endemic to a Wallacean ancient lake.

Heteroplasmy, the presence of multiple mitochondrial DNA (mtDNA) haplotypes within cells of an individual, is caused by mutation or paternal leakage. However, heteroplasmy is usually resolved to homoplasmy within a few generations because of germ-line bottlenecks; therefore, instances of heteroplasmy are limited in nature. Here, we report heteroplasmy in the ricefish species Oryzias matanensis, endemic to Lake Matano, an ancient lake in Sulawesi Island, in which one individual was known to have many heterozygous sites in the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. In this study, we cloned the ND2 gene for some additional individuals with heterozygous sites and demonstrated that they are truly heteroplasmic. Phylogenetic analysis revealed that the extra haplotype within the heteroplasmic O. matanensis individuals clustered with haplotypes of O. marmoratus, a congeneric species inhabiting adjacent lakes. This indicated that the heteroplasmy originated from paternal leakage due to interspecific hybridization. The extra haplotype was unique and contained two non-synonymous substitutions. These findings demonstrate that this hybridization-driven heteroplasmy was maintained across generations for a long time to the extent that the extra mitochondria evolved within the new host.

A study on the subchronic toxicity of triclocarban to the early-life development of oryzias melastigma and focused on the analysis of osmoregulatory regulation mechanisms.

Triclocarban (TCC), a novel antimicrobial agent found in personal care products, has been extensively detected in marine environments. However, research on the toxic effects of TCC on marine organisms remains inadequate. This study delved into the subchronic toxic effects of TCC on the early life stages of marine medaka (Oryzias melastigma, O. melastigma), revealing that TCC could reduce embryo heart rate and hatching rate while diminishing the survival rate of larvae. Biomarker assays indicated that TCC could inflict damage on the embryos' antioxidant and nervous systems. Transcriptomic analysis suggested that TCC could impact cell growth, reproduction, and various life processes, activating cancer signaling pathways, increasing the likelihood of cancer, and exerting toxic effects on the immune and osmoregulatory systems. To validate and enhance our understanding of TCC's unique toxic impact on the osmoregulatory system of O. melastigma, we conducted homology modeling and molecular docking analyses on the protein involved in osmoregulation. The study intuitively revealed the potential binding affinity of TCC to sodium/potassium-transporting ATPase subunit alph (ATP1A1), indicating its ability to disrupt osmotic balance in marine fish by affecting this target protein. In summary, the results of this study will further enhance our comprehension of the potential toxic effects and mechanisms of TCC on the early stages of marine fish, with a specific focus on its unique toxic effects in osmoregulation.

Day length regulates gonadotrope proliferation and reproduction via an intra-pituitary pathway in the model vertebrate Oryzias latipes.

In seasonally breeding mammals and birds, the production of the hormones that regulate reproduction (gonadotropins) is controlled by a complex pituitary-brain-pituitary pathway. Indeed, the pituitary thyroid-stimulating hormone (TSH) regulates gonadotropin expression in pituitary gonadotropes, via dio2-expressing tanycytes, hypothalamic Kisspeptin, RFamide-related peptide, and gonadotropin-releasing hormone neurons. However, in fish, how seasonal environmental signals influence gonadotropins remains unclear. In addition, the seasonal regulation of gonadotrope (gonadotropin-producing cell) proliferation in the pituitary is, to the best of our knowledge, not elucidated in any vertebrate group. Here, we show that in the vertebrate model Japanese medaka (Oryzias latipes), a long day seasonally breeding fish, photoperiod (daylength) not only regulates hormone production by the gonadotropes but also their proliferation. We also reveal an intra-pituitary pathway that regulates gonadotrope cell number and hormone production. In this pathway, Tsh regulates gonadotropes via folliculostellate cells within the pituitary. This study suggests the existence of an alternative regulatory mechanism of seasonal gonadotropin production in fish.

Deciphering the origin of developmental stability: The role of intracellular expression variability in evolutionary conservation.

Progress in evolutionary developmental biology (evo-devo) has deepened our understanding of how intrinsic properties of embryogenesis, along with natural selection and population genetics, shape phenotypic diversity. A focal point of recent empirical and theoretical research is the idea that highly developmentally stable phenotypes are more conserved in evolution. Previously, we demonstrated that in Japanese medaka (Oryzias latipes), embryonic stages and genes with high stability, estimated through whole-embryo RNA-seq, are highly conserved in subsequent generations. However, the precise origin of the stability of gene expression levels evaluated at the whole-embryo level remained unclear. Such stability could be attributed to two distinct sources: stable intracellular expression levels or spatially stable expression patterns. Here we demonstrate that stability observed in whole-embryo RNA-seq can be attributed to stability at the cellular level (low variability in gene expression at the cellular levels). We quantified the intercellular variations in expression levels and spatial gene expression patterns for seven key genes involved in patterning dorsoventral and rostrocaudal regions during early development in medaka. We evaluated intracellular variability by counting transcripts and found its significant correlation with variation observed in whole-embryo RNA-seq data. Conversely, variation in spatial gene expression patterns, assessed through intraindividual left-right asymmetry, showed no correlation. Given the previously reported correlation between stability and conservation of expression levels throughout embryogenesis, our findings suggest a potential general trend: the stability or instability of developmental systems-and the consequent evolutionary diversity-may be primarily anchored in intrinsic fundamental elements such as the variability of intracellular states.

Ran-GTP assembles a specialized spindle structure for accurate chromosome segregation in medaka early embryos.

Despite drastic cellular changes during cleavage, a mitotic spindle assembles in each blastomere to accurately segregate duplicated chromosomes. Mechanisms of mitotic spindle assembly have been extensively studied using small somatic cells. However, mechanisms of spindle assembly in large vertebrate embryos remain little understood. Here, we establish functional assay systems in medaka (Oryzias latipes) embryos by combining CRISPR knock-in with auxin-inducible degron technology. Live imaging reveals several unexpected features of microtubule organization and centrosome positioning that achieve rapid, accurate cleavage. Importantly, Ran-GTP assembles a dense microtubule network at the metaphase spindle center that is essential for chromosome segregation in early embryos. This unique spindle structure is remodeled into a typical short, somatic-like spindle after blastula stages, when Ran-GTP becomes dispensable for chromosome segregation. We propose that despite the presence of centrosomes, the chromosome-derived Ran-GTP pathway has essential roles in functional spindle assembly in large, rapidly dividing vertebrate early embryos, similar to acentrosomal spindle assembly in oocytes.

Loss of function in somatostatin receptor 5 has no impact on the growth of medaka fish due to compensation by the other paralogs.

Somatic growth in vertebrates is regulated endocrinologically by the somatotropic axis, headed by the growth hormone (GH) and the insulin growth factor-I (IGF-I). Somatostatin (Sst), a peptide hormone synthesized in the hypothalamus, modulates GH actions through its receptors (Sstr). Four Sstr subtypes (Sstr 1-3 and 5) have been identified in teleosts. However, little is known about whether they have a specific function or tissue expression. The aim of this study was to determine the role of sstr2 and sstr5 in the growth of the medaka (Oryzias latipes). The assessed expression pattern across diverse tissues highlighted greater prevalence of sstr1 and sstr3 in brain, intestine and muscle than in pituitary or liver. The expression of sstr2 was high in all the tissues tested, while sstr5 was predominantly expressed in the pituitary gland. A CRISPR/Cas9 sstr5 mutant with loss of function (sstr5-/-) was produced. Assessment of sstr5-/- indicated no significant difference with the wild type regarding growth parameters such as standard length, body depth, or peduncle depth. Furthermore, the functional loss of sstr5 had no impact on the response to a nutritional challenge. The fact that several sstr subtypes were upregulated in different tissues in sstr5-/- medaka suggests that in the mutant fish, there may be a compensatory effect on the different tissues, predominantly by sstr1 in the liver, brain and pituitary, with sstr2 being upregulated in pituitary and liver, and sstr3 only presenting differential expression in the brain. Analysis of the sstr subtype and the sstr5-/- fish showed that sstr5 was not the only somatostatin receptor responsible for Sst-mediated Gh regulation.

Trophic transfer of micro- and nanoplastics and toxicity induced by long-term exposure of nanoplastics along the rotifer (Brachionus plicatilis)-marine medaka (Oryzias melastigma) food chain.

Microplastics (MPs) and nanoplastics (NPs) are emerging pollutants in the ocean, but their transfer and toxicity along the food chains are unclear. In this study, a marine rotifer (Brachionus plicatilis)-marine medaka (Oryzias melastigma) food chain was constructed to evaluate the transfer of polystyrene MPs and NPs (70 nm, 500 nm, and 2 µm, 2000 µg/L) and toxicity of 70 nm PS-NPs (0, 20, 200, and 2000 µg/L) on marine medaka after long-term food chain exposure. The results showed that the amount of 70 nm NPs accumulated in marine medaka was 1.24 µg/mg, which was significantly higher than that of 500 nm NPs (0.87 µg/mg) and 2 µm MP (0.69 µg/mg). Long-term food chain exposure to NPs caused microflora dysbiosis, resulting in activation of toll-like receptor 4 (TLR4) pathway, which induced liver inflammation. Moreover, NPs food chain exposure increased liver and muscle tissue triglyceride and lactate content, but decreased the protein, sugar, and glycogen content. NPs food chain exposure impaired reproductive function and inhibited offspring early development, which might pose a threat to the sustainability of marine medaka population. Overall, the study revealed the transfer of MPs and NPs and the effects of NPs on marine medaka along the food chain.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

Comprehensive analysis of diel rhythmic expression of the medaka toll-like receptor gene family.

Several immune-related genes, including Toll-like receptors (TLR), are associated with circadian rhythms in mammals. However, information on the circadian rhythmic expression of TLRs in fish is limited. In this study, we aimed to analyze the regulation of diel oscillations in the expression of TLR genes in Japanese medaka (Oryzias latipes). The expression analysis revealed diel expression patterns of tlr1, tlr5m, tlr21, and clock genes (bmal1 and clock1) under a 12 h light:12 h dark cycle. The clock gene response element (E-box) was identified in the transcriptional regulatory regions of tlr1, tlr5m, and tlr21. Moreover, overexpressed bmal1 and clock1 enhanced expression levels of tlr1, tlr5m, and tlr21 in medaka embryo (OLHdrR-e3) cells. The expression of tlr1, tlr5m, and tlr21 was significantly decreased in OLHdrR-e3 after generating a bmal1 knockdown using a morpholino oligo. These results indicate the regulation of the diel rhythmic expression of several fish TLRs by clock genes.

Chemical and Visual Cues as Modulators of the Stress Response to Social Isolation in the Marine Medaka, Oryzias melastigma.

The marine medaka is emerging as a potential behavioral model organism for ocean studies, namely on marine ecotoxicology. However, not much is known on the behavior of the species and behavioral assays lack standardization. This study assesses the marine medaka as a potential model for chemical communication. We investigated how short exposure to visual and chemical cues mediated the stress response to social isolation with the light/dark preference test (LDPT) and the open field test (OFT). After a 5-day isolation period, and 1 h before testing, isolated fish were randomly assigned to one of four groups: (1) placed in visual contact with conspecifics; (2) exposed to a flow of holding water from a group of conspecifics; (3) exposed to both visual and chemical cues from conspecifics; or (4) not exposed to any stimuli (controls). During the LDPT, the distance traveled and transitions between zones were more pronounced in animals exposed to the conspecific's chemical stimuli. The time spent in each area did not differ between the groups, but a clear preference for the bright area in all animals indicates robust phototaxis. During the OFT, animals exposed only to chemical cues initially traveled more than those exposed to visual or both stimuli, and displayed lower thigmotaxis. Taken together, results show that chemical cues play a significant role in exploratory behavior in this species and confirm the LDPT and OFT as suitable tests for investigating chemical communication in this species.

Surface functional groups on nanoplastics delay the recovery of gut microbiota after combined exposure to sulfamethazine in marine medaka (Oryzias melastigma).

Nanoplastics can interact with antibiotics, altering their bioavailability and the ensuing toxicity in marine organisms. It is reported that plain polystyrene (PS) nanoplastics decrease the bioavailability and adverse effects of sulfamethazine (SMZ) on the gut microbiota in Oryzias melastigma. However, the influence of surface functional groups on the combined effects with SMZ remains largely unknown. In this study, adult O. melastigma were fed diet amended with 4.62 mg/g SMZ and 3.65 mg/g nanoplastics (i.e., plain PS, PS-COOH and PS-NH2) for 30 days (F0-E), followed by a depuration period of 21 days (F0-D). In addition, the eggs produced on the last day of exposure were cultured under standard protocols without further exposure for 2 months (F1 fish). The results showed that the alpha diversity or the bacterial community of gut microbiota did not differ among the SMZ + PS, SMZ + PS-COOH, and SMZ + PS-NH2 groups in the F0-E and F1 fish. Interestingly, during the depuration, a clear recovery of gut microbiota (e.g., increases in the alpha diversity, beneficial bacteria abundances and network complexity) was found in the SMZ + PS group, but not for the SMZ + PS-COOH and SMZ + PS-NH2 groups, indicating that PS-COOH and PS-NH2 could prolong the toxic effect of SMZ and hinder the recovery of gut microbiota. Compared to plain PS, lower egestion rates of PS-COOH and PS-NH2 were observed in O. melastigma. In addition, under the simulated fish digest conditions, the SMZ-loaded PS-NH2 was found to desorb more SMZ than the loaded PS and PS-COOH. These results suggested that the surface -COOH and -NH2 groups on PS could influence their egestion efficiency and the adsorption/desorption behavior with SMZ, resulting in a long-lasting SMZ stress in the gut during the depuration phase. Our findings highlight the complexity of the carrier effect and ecological risk of surface-charged nanoplastics and the interactions between nanoplastics and antibiotics in natural environments.

Effect of diisobutyl adipate on the expression of biomarker genes that respond to endocrine disruption and on gonadal sexual differentiation in Japanese medaka (Oryzias latipes).

Phthalate and non-phthalate plasticizers are used in polymer materials, such as plastic and rubber. It has recently been found that diisobutyl adipate (DIBA), which is considered an environmentally safe non-phthalate plasticizer, potentially acts as a thyroid disruptor in fish. Here, we investigated the sexual hormone effects of DIBA based on the expression levels of genes that respond to endocrine disruption and sexual hormone activity in the livers and gonads, and on gonadal sexual differentiation in Japanese medaka. Compared with the control group, the mRNA expression of chgH, vtg1, vtg2, and esr1 was significantly suppressed in the livers of DIBA exposed XX individuals. Furthermore, the mRNA expression of gsdf was significantly upregulated and downregulated in the gonads of XX and XY individuals, respectively. The mRNA expressions of esr1 and esr2b were significantly suppressed by DIBA exposure in the gonads of both XX and XY individuals. These observations suggest that DIBA has potential androgenic activity in Japanese medaka. However, normal testes and ovaries were observed in respective XY and XX medaka after DIBA exposure; therefore, these results suggest that DIBA may have weak androgenic activity.