Japanese Medaka Oryzias Latipes Research Articles

Novel Disinfection Byproducts Formed from the Pharmaceutical Gemfibrozil Are Bioaccumulative and Elicit Increased Toxicity Relative to the Parent Compound in Marine Polychaetes (Neanthes arenaceodentata)

Formation of halogenated disinfection byproducts (DBPs) from pharmaceutically active compounds has been observed in water supply systems following wastewater chlorination. Although research has been limited thus far, several studies have shown that halogenated DBPs may elicit increased toxicity compared to their parent compounds. For example, the lipid regulator gemfibrozil has been shown to form chlorogemfibrozil (Cl-gemfibrozil) and bromogemfibrozil (Br-gemfibrozil) following chlorination, which are more potent antiandrogens in male Japanese medaka (Oryzias latipes) compared to their parent compounds. In the present study, we aimed to characterize the bioaccumulative ability of halogenated gemfibrozil DBPs in marine polychaetes via chronic sediment exposures and, consequently, to assess the chronic and acute toxicity of halogenated gemfibrozil DBPs through sediment (in vivo) and aqueous (in vivo and in silico) toxicity evaluations. Following 28 day sediment exposures, Cl-gemfibrozil and Br-gemfibrozil bioaccumulated within Neanthes arenaceodentata, with both compounds reducing survival and growth. The biota-sediment accumulation factors determined for Cl-gemfibrozil and Br-gemfibrozil were 2.59 and 6.86, respectively. Furthermore, aqueous 96 h toxicity tests with N. arenaceodentata indicated that gemfibrozil DBPs elicited increased toxicity compared to the parent compound. While gemfibrozil had an acute LC50 value of 469.85 ± 0.096 mg/L, Cl-gemfibrozil and Br-gemfibrozil had LC50 values of 12.34 ± 0.085 and 9.54 ± 0.086 mg/L, respectively. Although acute toxicity is relatively low, our results indicate that halogenated gemfibrozil DBPs are bioaccumulative and may elicit effects in apex food web organisms prone to accumulation following lifelong exposures.

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.

ASC-deficiency impairs host defense against Aeromonas hydrophila infection in Japanese medaka, Oryzias latipes

Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a component of inflammasome, which plays crucial roles in the inflammatory response. In mammals, ASC regulates caspase-1 activation, thereby inducing pyroptosis and producing activated inflammatory cytokines. In addition, ASC also interacts with receptor-interacting protein kinase 2 (RIPK2) and induces nuclear factor-κB (NF-κB) activation. However, the role of ASC remains poorly understood in fish. In this study, we focused on elucidating the role of ASC in fish that were infected with Aeromonas hydrophila using Japanese medaka (Oryzias latipes) as fish model, and ASC-knockout (KO) medaka was established using CRISPR-Cas9 system. ASC-KO and wild type (WT) medakas were infected with A. hydrophila, and mortality was observed. ASC-KO medaka demonstrated higher mortality than WT. Moreover, the expression of immune-related genes in the kidney and intestine of the ASC-KO and WT medakas challenged with A. hydrophila were analyzed. Following A. hydrophila infection, the kidney of ASC-KO medaka exhibited significantly lower expression of NF-κB regulated genes (e.g., IL-1β, IL-6, IL-8 and TNF-α) and RIPK2 gene than in WT kidney. Moreover, to investigate the immune response against A. hydrophila via ASC in the medaka, bacterial burden, superoxide anion production, and lactate dehydrogenase release in the kidney cells of ASC-KO medaka were measured. After infection, these responses in ASC-KO medaka were significantly decreased compared to those in WT. These results suggest that the medaka ASC plays a critical role against A. hydrophila infection by inducing inflammatory responses and cell death for bacterial clearance.

Assessment of reproductive and developmental effects of graphene oxide on Japanese medaka (Oryzias latipes)

Due to its unique properties, graphene oxide (GO) has potential for biomedical and electronic applications, however environmental contamination including aquatic ecosystem is inevitable. Moreover, potential risks of GO in aquatic life are inadequately explored. Present study was designed to evaluate GO as an endocrine disrupting chemical (EDC) using the model Japanese medaka (Oryzias latipes). GO was injected intraperitoneally (25–200 μg/g) once to breeding pairs and continued pair breeding an additional 21 days. Eggs laid were analyzed for fecundity and the fertilized eggs were evaluated for developmental abnormalities including hatching. Histopathological evaluation of gonads, liver, and kidneys was made 21 days post-injection. LD50 was found to be sex-dependent. Fecundity tended to reduce in a dose-dependent manner during early post-injection days; however, the overall evaluation showed no significant difference. The hatchability of embryos was reduced significantly in the 200 μg/g group; edema (yolk and cardiovascular) and embryo-mortality remained unaltered. Histopathological assessment identified black particles, probably agglomerated GO, in the gonads of GO-treated fish. However, folliculogenesis in stromal compartments of ovary and the composition of germinal elements in testis remained almost unaltered. Moreover, granulosa and Leydig cells morphology did not indicate any significant EDC-related effects. Although liver and kidney histopathology did not show GO as an EDC, some GO-treated fish accumulated proteinaceous fluid in hepatic vessels and induced hyperplasia in interstitial lymphoid cells (HIL) located in kidneys. GO agglomerated in medaka gonads after 21-days post-injection. However, gonad histopathology including granulosa and Leydig cells alterations were associated with GO toxicity rather than EDC effects.

Energy and nitrogenous waste from glutamate/glutamine catabolism facilitates acute osmotic adjustment in non-neuroectodermal branchial cells

Maintenance of homeostasis is one of the most important physiological responses for animals upon osmotic perturbations. Ionocytes of branchial epithelia are the major cell types responsible for active ion transport, which is mediated by energy-consuming ion pumps (e.g., Na+-K+-ATPase, NKA) and secondary active transporters. Consequently, in addition to osmolyte adjustments, sufficient and immediate energy replenishment is essenttableial for acclimation to osmotic changes. In this study, we propose that glutamate/glutamine catabolism and trans-epithelial transport of nitrogenous waste may aid euryhaline teleosts Japanese medaka (Oryzias latipes) during acclimation to osmotic changes. Glutamate family amino acid contents in gills were increased by hyperosmotic challenge along an acclimation period of 72 hours. This change in amino acids was accompanied by a stimulation of putative glutamate/glutamine transporters (Eaats, Sat) and synthesis enzymes (Gls, Glul) that participate in regulating glutamate/glutamine cycling in branchial epithelia during acclimation to hyperosmotic conditions. In situ hybridization of glutaminase and glutamine synthetase in combination with immunocytochemistry demonstrate a partial colocalization of olgls1a and olgls2 but not olglul with Na+/K+-ATPase-rich ionocytes. Also for the glutamate and glutamine transporters colocalization with ionocytes was found for oleaat1, oleaat3, and olslc38a4, but not oleaat2. Morpholino knock-down of Sat decreased Na+ flux from the larval epithelium, demonstrating the importance of glutamate/glutamine transport in osmotic regulation. In addition to its role as an energy substrate, glutamate deamination produces NH4+, which may contribute to osmolyte production; genes encoding components of the urea production cycle, including carbamoyl phosphate synthetase (CPS) and ornithine transcarbamylase (OTC), were upregulated under hyperosmotic challenges. Based on these findings the present work demonstrates that the glutamate/glutamine cycle and subsequent transepithelial transport of nitrogenous waste in branchial epithelia represents an essential component for the maintenance of ionic homeostasis under a hyperosmotic challenge.

Occurrences of benzalkonium chloride in streams near a pharmaceutical manufacturing complex in Korea and associated ecological risk

Benzalkonium chloride (BKC) is a commonly used preservative in personal care products and pharmaceutical preparations. However, its ecological risks are not well understood because of lack of monitoring data and ecotoxicological information. In the present study, occurrence of BKC was investigated in the waters near a pharmaceutical manufacturing complex of South Korea and its acute and chronic ecotoxicities were evaluated using Daphnia magna and Japanese medaka (Oryzias latipes). Associated ecological risks were estimated by calculating hazard quotients (HQs). In addition, endocrine disruption potency of BKC was compared with those of other frequently used preservatives using human adrenal (H295R) and rat pituitary (GH3) cells. High concentration of BKC was detected at locations near the pharmaceutical manufacturing plants, i.e., 35.8 μg/L for dodecyl benzyl dimethyl ammonium chloride (BKC–C12), and 21.6 μg/L tetradecyl benzyl dimethyl ammonium chloride (BKC–C14). In Daphnia, 48 h immobilization EC50 and 21 d reproduction NOEC were determined at 41.1 μg/L and ≥10.8 μg/L, respectively. For O. latipes, 96 h LC50 was determined at 246 μg/L while the growth inhibition NOEC was ≥113.4 μg/L following early life stage exposure. BKC significantly up-regulated vitellogenin gene of juvenile fish, indicating its endocrine disrupting potential in fish. Exposure to BKC increased steroid hormone level in H295R cells, and induced cytotoxicity in GH3 cells. HQ values of BKC were determined at greater than one in the ambient water near pharmaceutical manufacturing facilities. Considering high ecological risk and endocrine disrupting potential, long-term consequences of BKC contamination in aquatic ecosystem need to be examined.

Transcriptional responses in newly-hatched Japanese medaka (Oryzias latipes) associated with developmental malformations following diluted bitumen exposure

Japanese medaka embryos were exposed to water accommodated fractions (WAF) and chemically-enhanced WAF of two types of diluted bitumen (dilbit) at concentrations bracketing the EC50s for developmental malformations. Within these treatments, fish were grouped based on the presence or absence of developmental malformations (e.g., blue sac disease (BSD)), and analyzed for novel transcriptomic responses. Microarray analyses identified novel biomarkers and gene networks in dilbit-exposed malformed embryos that were not evident in dilbit-exposed fish without BSD or in controls without dilbit. The top differentially expressed genes (DEGs) included cytochrome P450 transcripts (cyp1) in fish from all dilbit treatments (malformed and non-malformed fish), as well as: fibroblast growth factor (fgf7), AHR repressor (ahrr), and squalene monooxygenase (sqle). In dilbit-exposed fish that did not develop BSD, the only reported individual DEG was eukaryotic translation initiation factor 3 subunit D (eif3d). However, a number of other pathways were enriched, including melatonin effects on circadian clock and the antioxidant response, estrogen and androgen metabolism as well as many receptor signaling pathways. Pathways associated with hedgehog, steroid biosynthesis, and Wnt signaling were significantly altered between low and high concentrations of dilbit exposure. An effect of the dispersant control on swim bladder development was observed at concentrations 10-fold higher than those used to disperse dilbit, and a number of gene targets unique to fish in this comparison were affected. This suggests that the toxic effects of dispersant may involve alternative mechanisms to dilbit, but cause similar phenotypic responses. This study identified novel biomarkers in fish exposed to dilbit, with or without visual malformations, that can be used to assess the risks of dilbit to aquatic ecosystem health.

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.

The Effects of 17α-Ethinylestradiol on the Heart Rate of Embryonic Japanese Medaka (Oryzias latipes).

Estrogen toxicity has been an area of priority in aquatic toxicology over the last 20 yr. Currently, estrogen toxicity is primarily linked to classical estrogen signaling, the interaction of estrogen receptors alpha and beta (ERα and ERβ). Recent evidence has indicated that a rapid, nongenomic, nonclassical estrogen signaling pathway exists via the G protein-coupled estrogen receptor (GPER), which is expressed in many biological systems, with roles in the cardiovascular system. The objective of the present study was to investigate the effect of 17α-ethinylestradiol (EE2) on the heart rate of embryonic Japanese medaka (Oryzias latipes). A significant decrease (bradycardia) in embryonic heart rate was observed at all treatment concentrations (0.1, 1, 10, 100, and 1000 ng/L EE2) at 144, 168, and 192 h postfertilization (hpf; p ≤ 0.05), whereas 120 and 216 hpf embryos experienced a significant decrease from the control at 10, 100, and 1000 ng/L EE2 and 0.1, 100, and 1000 ng/L EE2, respectively (p ≤ 0.05). In addition, using select estrogen receptor modulators, it was demonstrated that estrogen-induced bradycardia appears to be linked to GPER and not ERα and ERβ. The present study highlights GPER as a novel and alternative mode of action for EE2 toxicity at environmentally relevant concentrations. Environ Toxicol Chem 2020;39:904-912. © 2020 SETAC.

Interleukin-17A/F1 Deficiency Reduces Antimicrobial Gene Expression and Contributes to Microbiome Alterations in Intestines of Japanese medaka (Oryzias latipes).

In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka (Oryzias latipes) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1β (IL-1β), complement 1q subunit C (C1qc), transferrin a (Tfa), and G-type lysozyme (LyzG), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB (pla2g1b), and elastase-1-like (CELA1), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the intestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome.

Novel Nile tilapia Neu1 sialidases: Molecular cloning and biochemical characterization of the sialidases Neu1a and Neu1b

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.

Chronic microfiber exposure in adult Japanese medaka (Oryzias latipes).

Microplastic fibers (MFs) pollute aquatic habitats globally via sewage release, stormwater runoff, or atmospheric deposition. Of the synthetic MFs, polyester (PES) and polypropylene (PP) are the most common. Field studies show that fish ingest large quantities of MFs. However, few laboratory studies have addressed host responses, particularly at the organ and tissue levels. Adult Japanese medaka (Oryzias latipes), a laboratory model fish, were exposed to aqueous concentrations of PES or PP MFs (10,000 MFs/L) for 21 days. Medaka egested 1,367 ± 819 PES MFs (0.1 ± 0.04 mg) and 157 ± 105 PP MFs (1.4 ± 0.06 mg) per 24 hrs, with PP egestion increasing over time. Exposure did not result in changes in body condition, gonadosomatic- or hepatosomatic indices. PES exposure resulted in no reproductive changes, but females exposed to PP MFs produced more eggs over time. MF exposure did not affect embryonic mortality, development, or hatching. Scanning electron microscopy (SEM) of gills revealed denuding of epithelium on arches, fusion of primary lamellae, and increased mucus. Histologic sections revealed aneurysms in secondary lamellae, epithelial lifting, and swellings of inner opercular membrane that altered morphology of rostral most gill lamellae. SEM and histochemical analyses showed increased mucous cells and secretions on epithelium of foregut; however, overt abrasions with sloughing of cells were absent. For these reasons, increased focus at the tissue and cell levels proved necessary to appreciate toxicity associated with MFs.

3-(4-Methylbenzylidene) camphor induced reproduction toxicity and antiandrogenicity in Japanese medaka (Oryzias latipes)

To assess the toxic effects of 3-(4-Methylbenzylidene) camphor (4-MBC) at environmentally relevant concentrations on the reproduction and development of Japanese medaka (Oryzias latipes), adult paired medaka (F0) were exposed to 5, 50, and 500 μg/L 4-MBC for 28 d in the current study. The fecundity and fertility were significantly decreased at 500 μg/L 4-MBC (p < 0.05). Histological observations showed that spermatogenesis in F0 males was significantly inhibited at 50 and 500 μg/L 4-MBC, similar to the effects obtained with all treatments of plasma 11-ketotestosterone (p < 0.05). Moreover, the plasma vitellogenin and estradiol levels in F0 females were significantly increased at 5 μg/L 4-MBC (p < 0.05). All the transcripts of hypothalamic-pituitary-gonadal (HPG) axis-related genes tested in the brains and gonads of males were significantly increased at all treatments, similar to the effects obtained for erα, erβ and vtg in the livers and in contrast to those found for arα in the livers (p < 0.05). Equal numbers of embryos were exposed to tap water and 4-MBC solutions. Significantly increased times to hatching, decreased hatching rates and decreased body lengths at 14-day post-hatching (dph) were obtained at 500 μg/L 4-MBC treatment (p < 0.05). The cumulative death rates at 14 dph were significantly increased with all the treatments (p < 0.05). Therefore, our results showed that long-term exposure to 50 and 500 μg/L 4-MBC causes reproductive and developmental toxicity and thus provide new insight into antiandrogenicity and the mechanism of 4-MBC in Japanese medaka.

Reproductive toxicity and estrogen activity in Japanese medaka (Oryzias latipes) exposed to environmentally relevant concentrations of octocrylene

The growing use of octocrylene (OC) in sunscreens has posed a great threat to aquatic organisms. In the present study, to assess its reproductive toxicity and mechanism, paired Japanese medaka (Oryzias latipes) (F0) were exposed to OC at nominal concentrations of 5, 50, and 500 μg/L for 28 d. Significant increases were observed in the gonadosomatic index (GSI) and hepatosomatic index (HSI) of F0 medaka at 500 μg/L OC (p < 0.05) without significant differences in fecundity. The fertility was significantly decreased at all treatments (p < 0.05). Significant increases in the percent of mature oocytes were observed at 5 and 500 μg/L OC, in which contrary to the percent of spermatozoa (p < 0.05). The plasma sex hormones and vitellogenin levels significantly increased in males at all treatments and in females at 50 and 500 μg/L OC (p < 0.05). In addition, the levels of fshβ and lhβ in the brains and the levels of fshr, lhr and cyp17α in the gonads were significantly upregulated in males at all treatments (p < 0.05), in line with those of ar, erα, erβ and cyp19β in the brains of male and female. The upregulation of vtg in male and female livers was observed only at 500 μg/L OC and upregulation of star and hsd3β was observed in testis at all treatments (p < 0.05). Continued exposure to OC significantly induced increases in the time to hatching, morphological abnormality rates, and cumulative death rates of F1 embryos, inconsistent with body length of F1 larvae (p < 0.05). Therefore, the responses of the exposed fish at the biochemical and molecular levels indicated reproductive toxicity and estrogenic activity of OC, providing insights into the mechanism of OC.

Bisphenol A induces a shift in sex differentiation gene expression with testis-ova or sex reversal in Japanese medaka (Oryzias latipes).

Bisphenol A (BPA), a very important raw material in the plastics industry, is an endocrine-disrupting chemical in teleost fish. Although BPA induces testis-ova and sex reversal in teleost fish species, the molecular mechanism remains unclear. We evaluated the effects of BPA (measured concentrations: 45, 92, 326, 1030 and 3406 μg/L) on Japanese medaka (Oryzias latipes) using OECD TG234 (2011, Fish Sexual Development Test, OECD Guidelines for the Testing of Chemicals, Section 2). BPA at 1030 and 3406 μg/L induced testis-ova and sex reversal with female-type secondary sexual characteristics in XY males at 30 and 60 days posthatching (dph). Then we examined the BPA effect on the expression of sex differentiation genes related to the testis-ova and sex reversal in XY medaka. BPA exposure (1030 and 3406 μg/L) suppressed gsdf mRNA expression and increased cyp19a1a mRNA expression in XY individuals at stage 38 and 30 dph, although foxl2 mRNA expression showed no change. Interestingly, the concentration of BPA that suppressed gsdf mRNA expression at the larval stage was consistent with that needed to induce testis-ova and sex reversal. These results suggest that the gsdf gene at the embryonic stage can be used as a useful biomarker for predicting the impact of estrogenic endocrine-disrupting chemicals on sexual differentiation in Japanese medaka.

Chronic dietary exposure to polystyrene microplastics in maturing Japanese medaka (Oryzias latipes)

Fish studies report consumption of microplastics (MPs) in the field, and concern exists over associated risks. However, laboratory studies with adult fish are scarce. In this study, outbred and see-through Japanese medaka (Oryzias latipes) were fed diets amended with 500, 1000, or 2000 μg/g 10 μm fluorescent spherical polystyrene microplastics (MPs) for 10 weeks during their maturation from juveniles to spawning adults. No behavioral changes, growth differences, or mortalities occurred. In vivo examinations and histologic sections showed no evidence of translocation of MPs from the gut to other internal organs. Mature females experienced dose-dependent decreases in egg number. Scanning electron microscopic examination of gills and gut revealed MPs in both areas. Swollen enterocytes were observed on apices of gut folds only in exposed fish. These were particularly apparent in foreguts of the high exposure group. Enterocytes with eroded brush borders were found in foregut of high and medium exposure groups. Increased mucus production, in long strands and sheets, was seen over primary and secondary lamellae of gills. Histological analysis showed alteration in buccal cavity, kidney, and spleen. Thickening and roughening of epithelium in headgut and pharynx and cellular alterations in spleen occurred. Head kidney was the primary site of alteration. Glomerulopathy and nephrogenesis were observed in exposed fish, increasing in severity with exposure level.

ZEB316: A Small Stand-Alone Housing System to Study Microplastics in Small Teleosts.

Many anthropogenic chemicals and plastic debris end up in the aquatic ecosystem worldwide, representing a major concern for the environment and human health. Small teleosts, such as zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes), offer significant advantages over classical animal models and are currently used as first-line organisms to assess environmental risks associated with many aquatic toxicants. Toxicological studies require the use of inert materials and controlled conditions. Yet, none of the available commercialized systems is adequate to assess the toxic effect of microplastics, because they contain components made of plastic polymers that may release micrometric plastic particles, leach manufacturing compounds, or adsorb chemicals. The ZEB316 stand-alone housing system presented in this study is meant to be a cost-effective and easy-to-built solution to perform state-of-the-art toxicological studies. It is built with inert and corrosion-resistant materials and provides good housing conditions through efficient recirculation and filtration systems. Assessment of water parameters and fish growth performance showed that the ZEB316 provides housing conditions comparable to those available from commercial housing systems.

Selenomethionine exposure affects chondrogenic differentiation and bone formation in Japanese medaka (Oryzias latipes)

Excess selenium entering the aquatic environment from anthropogenic activities has been associated with developmental abnormalities in fish including skeletal deformities of the head and spine. However, mechanisms of this developmental toxicity have not been well-characterized. In this study, Japanese medaka (Oryzias latipes) embryos were exposed to seleno-l-methionine (Se-Met) in a range of concentrations. Gene expression was evaluated for sex-determining region Y (SRY)-related box (Sox9a and Sox9b), runt-related transcription factor 2 (Runx2), and melatonin receptor (Mtr). Alterations in the length of Meckel’s cartilage, tail curvature, and decreased calcification were observed in skeletal stains at 10- and 22-days post-fertilization (dpf). Embryonic exposure of Osterix-mCherry transgenic medaka resulted in fewer teeth. Sox9a and Sox9b were up-regulated, while Runx2 and Mtr were down-regulated by Se-Met prior to hatch. Whole mount in situ hybridization (WISH) localized gene expression to areas observed to be affected in vivo. In addition, Se-Met exposures of a Mtr morpholino (Mtr-MO) as well as Luzindole exposed embryos developed similar skeletal malformations, supporting involvement of Mtr. These findings demonstrate that Se-Met modulates expression of key genes involved in chondrogenic differentiation and bone formation during development.

Molecular characterization and expression of two interleukin-17 receptor A genes on different chromosomes in Japanese medaka, Oryzias latipes.

In mammals, interleukin 17 (IL-17), which is produced mainly by Th17 cells, is a hallmark inflammatory cytokine that plays key roles in the protection against infection and intestinal mucosal immunity. The mammalian IL-17 receptor family comprises five members (IL-17RA-E). Of these, IL-17RA is important in the control of the bacterial microbiota in mucosal tissues, particularly in the intestine, where it acts as a receptor for IL-17A and -F. In this study, the nucleotide sequence of IL-17RA1 cDNA from Japanese medaka (Oryzias latipes) of the Cab strain was determined and compared to two IL-17RA cDNAs (i.e., IL-17RA1 and IL-17RA2) of Japanese medaka Hd-rR strain downloaded from NCBI. Hd-rR 17RA1 and IL-17RA2 were located on chromosome 23 and chromosome 6, respectively, and phylogenetic tree analysis revealed that teleost IL-17RA1 and IL-17RA2 were separated in different clusters. Synteny analysis revealed that Japanese medaka IL-17RA1 and mammalian IL-17RA are conserved. IL-17RA1 expression levels in the gills, intestine, whole kidney, skin, and spleen were significantly higher than those of IL-17RA2, suggesting that IL-17RA1 is an important functional receptor in mucosal immunity. Interestingly, the expression levels of both IL-17RA genes were notably higher in the posterior than in the anterior intestinal tract section. Furthermore, despite its lower basal expression, IL-17RA2 expression was significantly increased at 72 h post Edwardsiella tarda infection.

Toxic effects of fluridone on early developmental stages of Japanese Medaka (Oryzias latipes)

The herbicide fluridone is intensively applied to control invasive aquatic plants globally, including in the Sacramento and San Joaquin Delta (the Delta), California, USA. Our previous study revealed that the adult stage of Delta Smelt showed acute and sub-lethal adverse effects following 6 h of exposure to environmentally relevant concentrations of fluridone. To further investigate mechanisms of toxicity of fluridone and to assess its toxicity to early life stages of fish, we performed additional exposures using the fish model Japanese Medaka (Oryzias latipes). Male and female Medaka embryos were exposed to concentrations of fluridone for 14 d and showed reduced hatching success in a dose dependent manner. The half maximal effective concentration for the hatching success was 2.3 mg L−1. In addition, male and female Medaka larvae were acute exposed to fluridone for 6 h to assess their swimming behavior and gene expression patterns. Fish exposed to fluridone at 4.2 mg L−1 or higher became lethargic and showed abnormal swimming behavior. The response to the stimuli was significantly impaired by fluridone at 21 mg L−1 and above in males, and at 104 mg L−1 in females. Transcriptome analysis identified a total of 799 genes that were significantly differentially expressed, comprising 555 up-regulated and 244 down-regulated genes in males exposed to 21 mg L−1 of fluridone. The gene set enrichment analysis indicated a number of biological processes altered by fluridone. Among the genes involved in those biological processes, the expression of the genes, acetylcholinesterase, retinoic acid receptor, insulin receptor substrate, glutathione reductase, and glutathione S transferase, exhibited dose- and sex-dependent responses to fluridone. The study indicated that fluridone exposure led to detrimental toxic effects at early developmental stages of fish, by disturbing the biological processes of growth and development, and the nervous system, inducing oxidative stress and endocrine disruption.