Electric organ discharge from electric eel facilitates DNA transformation into teleost larvae in laboratory conditions.

Background: Electric eels (Electrophorus sp.) are known for their ability to produce electric organ discharge (EOD) reaching voltages of up to 860 V. Given that gene transfer via intense electrical pulses is a well-established technique in genetic engineering, we hypothesized that electric eels could potentially function as a gene transfer mechanism in their aquatic environment. Methods: To investigate this hypothesis, we immersed zebrafish larvae in water containing DNA encoding the green fluorescent protein (GFP) and exposed them to electric eel's EOD. Results and Discussion: Some embryos exhibited a mosaic expression of green fluorescence, in contrast to the control group without electrical stimulation, which showed little distinct fluorescence. This suggests that electric eel EOD has the potential to function as an electroporator for the transfer of DNA into eukaryotic cells. While electric eel EOD is primarily associated with behaviors related to sensing, predation, and defense, it may incidentally serve as a possible mechanism for gene transfer in natural environment. This investigation represents the initial exploration of the uncharted impact of electric eel EOD, but it does not directly establish its significance within the natural environment. Further research is required to understand the ecological implications of this phenomenon.

Wnt4a Is Indispensable for Genital Duct Elongation but Not for Gonadal Sex Differentiation in the Medaka, Oryzias latipes.

In most vertebrates, the oviducts and sperm ducts are derived from the Müllerian ducts and Wolffian ducts, respectively. However, in teleosts, the genital ducts are formed by the posterior extension of gonads in both sexes. Whether the genital ducts of teleosts are newly evolved organs or variants of Müllerian ducts is an important question for understanding evolutionary mechanisms of morphogenesis. One of the genes essential for Müllerian duct formation in mice is Wnt4, which is expressed in the mesenchyme and induces invagination of the coelomic epithelium and its posterior elongation. Here, we addressed the above question by examining genital duct development in mutants of two Wnt4 genes in the medaka (wnt4a is orthologous to mouse Wnt4, and wnt4b is paralogous). The wnt4b mutants had a short body but were fertile with normal genital ducts. In contrast, both male and female wnt4a mutants had their posterior elongation of the gonads stopped within or just outside the coelom. The mutants retained the posterior parts of ovarian cavities or sperm duct primordia, which are potential target tissues of Wnt4a. The gonads of female scl mutants (unable to synthesize sex steroids) lacked these tissues and did not develop genital ducts. Medaka wnt4a was expressed in the mesenchyme ventral to the genital ducts in both sexes. Taken together, the data strongly suggest that the mouse Müllerian ducts and the medaka genital ducts share homologous developmental processes. Additionally, the wnt4a or wnt4b single mutants and the double mutants did not show sex-reversal, implying that both genes are dispensable for gonadal sex differentiation in the medaka.

Establishment of transgenic epithelium-specific Cre-recombinase driving medaka (Oryzias latipes) by homology repair mediated knock-in.

Deletion of gene expression in the target tissues and cells is an effective strategy for elucidating the physiological functions of the protein of interest. For tissue-specific and/or inducible gene deletion, the Cre-loxP system has been widely used in various model organisms including medaka (Oryzias latipes). The epithelium is the key tissue, locating at the outermost area and playing a role in barrier to external stimuli. Despite a large genetic toolbox developed in medaka, there is no available Cre-driver line that works in an epithelium-specific manner. Here, we established epithelium-specific Cre-driver lines in medaka using a homology-directed repair mediated knock-in approach with CRISPR/Cas9, targeting each of periplakin and keratin genes. We show that Cre-recombinase is expressed exclusively in the epithelium in the knock-in lines and that it efficiently and specifically induces recombination in the tissues. These Cre-driver lines are useful for studying the functions of proteins expressed in the epithelium.

Pigmentation and TYRP1 expression are mediated by zinc through the early secretory pathway-resident ZNT proteins.

Tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are essential for pigmentation. They are generally classified as type-3 copper proteins, with binuclear copper active sites. Although there is experimental evidence for a copper cofactor in TYR, delivered via the copper transporter, ATP7A, the presence of copper in TYRP1 and TYRP2 has not been demonstrated. Here, we report that the expression and function of TYRP1 requires zinc, mediated by ZNT5-ZNT6 heterodimers (ZNT5-6) or ZNT7-ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in hypopigmentation in medaka fish and human melanoma cells, and is accompanied by immature melanosomes and reduced melanin content, as observed in TYRP1 dysfunction. The requirement of ZNT5-6 and ZNT7 for TYRP1 expression is conserved in human, mouse, and chicken orthologs. Our results provide novel insights into the pigmentation process and address questions regarding metalation in tyrosinase protein family.

Genome Editing of Medaka.

Medaka (Oryzias latipes), along with zebrafish (Danio rerio), is a useful experimental model fish. Here, we describe a simple method for generating medaka gene knockout strains using an automated microchip electrophoresis system. We also describe a method for targeted gene knockin using a plasmid carrying a sequence that does not cause off-target effects in medaka. Additionally, knockin method without plasmid cloning is described.

Cholesterol accumulation in ovarian follicles causes ovulation defects in Abca1a -/- Japanese medaka (Oryzias latipes).

ATP-binding cassette A1 (ABCA1) is a membrane protein, which exports excess cellular cholesterol to generate HDL to reduce the risk of the onset of cardiovascular diseases (CVD). In addition, ABCA1 exerts pleiotropic effects on such as inflammation, tissue repair, and cell proliferation and migration. In this study, we explored the novel physiological roles of ABCA1 using Japanese medaka (Oryzias latipes), a small teleost fish. Three Abca1 genes were found in the medaka genome. ABCA1A and ABCA1C exported cholesterol to generate nascent HDL as human ABCA1 when expressed in HEK293 cells. To investigate their physiological roles, each Abca1-deficient fish was generated using the CRISPR-Cas9 system. Abca1a -/- female medaka was found to be infertile, while Abca1b -/- and Abca1c -/- female medaka were fertile. In vitro ovarian follicle culture suggested that Abca1a deficiency causes ovulation defects. In the ovary, ABCA1A was expressed in theca cells, an outermost layer of the ovarian follicle. Total cholesterol content of Abca1a -/- ovary was significantly higher than that of the wild-type, while estrogen and progestin contents were compatible with those of the wild-type. Furthermore, cholesterol loading to the wild-type follicles caused ovulation defects. These results suggest that ABCA1A in theca cells regulates cholesterol content in the ovarian follicles and its deficiency inhibits successful ovulation through cholesterol accumulation in the ovarian follicle.

LAT1 expression influences Paneth cell number and tumor development in ApcMin/+ mice.

BACKGROUND: Amino acid transporters play an important role in supplying nutrition to cells and are associated with cell proliferation. L-type amino acid transporter 1 (LAT1) is highly expressed in many types of cancers and promotes tumor growth; however, how LAT1 affects tumor development is not fully understood. METHODS: To investigate the role of LAT1 in intestinal tumorigenesis, mice carrying LAT1 floxed alleles that also expressed Cre recombinase from the promoter of gene encoding Villin were crossed to an ApcMin/+ background (LAT1fl/fl; vil-cre; ApcMin/+), which were subject to analysis; organoids derived from those mice were also analyzed. RESULTS: This study showed that LAT1 was constitutively expressed in normal crypt base cells, and its conditional deletion in the intestinal epithelium resulted in fewer Paneth cells. LAT1 deletion reduced tumor size and number in the small intestine of ApcMin/+ mice. Organoids derived from LAT1-deleted ApcMin/+ intestinal crypts displayed fewer spherical organoids with reduced Wnt/ß-catenin target gene expression, suggesting a low tumor-initiation capacity. Wnt3 expression was decreased in the absence of LAT1 in the intestinal epithelium, suggesting that loss of Paneth cells due to LAT1 deficiency reduced the risk of tumor initiation by decreasing Wnt3 production. CONCLUSIONS: LAT1 affects intestinal tumor development in a cell-extrinsic manner through reduced Wnt3 expression in Paneth cells. Our findings may partly explain how nutrient availability can affect the risk of tumor development in the intestines.

Targeted deletion of ecto-5'-nucleotidase results in retention of inosine monophosphate content in postmortem muscle of medaka (Oryzias latipes).

Inosine monophosphate (IMP) is an important indicator of meat freshness and contributes to its umami taste. An attractive strategy for enhancing umami is to suppress the IMP-degrading activity and increase the IMP content in the skeletal muscle through genome editing technology using the CRISPR-Cas9 system. However, the molecular mechanisms underlying IMP degradation remain unclear. We cloned two ecto-5'-nucleotidase genes, designated as ecto-5'-nucleotidase-a (nt5ea) and ecto-5'-nucleotidase-b (nt5eb), from medaka (Oryzias latipes), a vertebrate model organism. Expression analysis using embryos showed that nt5ea or nt5eb overexpression remarkably upregulated IMP degradation, and that the IMP-degrading activity was higher in Nt5ea than in Nt5eb. Furthermore, we established frame-shifted or large deletion (lacking nt5ea or nt5eb locus) mutant strains and assayed the effects of gene disruptions on the amount of IMP in skeletal muscle. The nt5ea-deficient medaka showed considerable higher levels of IMP at 48 h postmortem than did the wild-type fish. The nt5eb mutants also exhibited higher IMP contents than that in the wild types, but the increase was less than that in the nt5ea mutants. Our results demonstrated that nt5e is an important regulator of IMP levels in skeletal muscle and that its loss of function was effective in maintaining IMP content.

Constitution of mucosa-associated microbiota in the lower digestive tract does not change in early stage of non-alcoholic fatty liver disease with fecal dysbiosis.

Background and Aim: Regarding the gut-liver axis, fecal dysbiosis is implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The significance of mucosa-associated microbiota (MAM, which is present in the mucin layer covering the intestinal mucosa) has not been well explored. We aimed to clarify the characteristics of MAM in patients with NAFLD. Methods: MAM were obtained from seven patients with early-stage NAFLD and seven controls by colonoscopy in five locations (terminal ileum, cecum, ascending and sigmoid colon, and rectum) using mucosal brushes. The microbial 16S rDNA profiles of the MAM and fecal microbiota of patients in the NAFLD and control groups were analyzed. Results: α-diversities of fecal microbiota were decreased in patients with NAFLD (observed species, Shannon index, and Chao1: 174.57 vs 134.86, 5.51 vs 4.65, and 206.34 vs 167.91; P = 0.048, 0.067, and 0.087, respectively), and microbial composition analyses by principal coordinate analysis differed between the fecal microbiota of patients with NAFLD and those of controls (permutational analysis of variance [PERMANOVA] of weighted and unweighted: Pseud-F: 1.4179/P-value: 0.05 and Pseud-F: 2.1497/P-value: 0.049, respectively). However, α-diversities or microbial composition of MAM in most parts of the intestine did not differ significantly between the NAFLD and control groups. Unclassified Rikenellaceae, Oscillospira, Odoribacter, unclassified clostridiales, and Holdemania were decreased in the feces of patients with NAFLD (determined by linear discriminant analysis effect size), but five (except Holdemania) of the six genera were not decreased in the MAM of these patients. Conclusion: In early-stage NAFLD, MAM was uniform and relatively stable throughout the intestine, even when fecal dysbiosis appeared.

Medaka, Oryzias latipes, egg envelopes are created by ovarian-expressed ZP proteins and liver-expressed choriogenins.

The medaka (Oryzias latipes) egg envelope (chorion) is composed of three major glycoproteins, Zona Interna (ZI)-1, -2, and -3, that originate in the spawning female liver as the precursor proteins Choriogenin (Chg.)H, Chg.Hm, and Chg.L, respectively. These ZI and Chg. proteins contain a structural ZP protein domain that is conserved among the egg envelope proteins of all animals. While ovarian expression of ZP proteins (e.g., ZPCs and ZPB) has been reported in medakas, the functions of these proteins remain unknown. Thus, the present study aimed to determine whether the ovary-expressed medaka ZP protein, mZPC5, is involved in forming the chorion matrix.The mZPC5 gene (mzpc5) was expressed in the ovaries but not the livers of mature female medakas, as shown by reverse transcription-polymerase chain reaction assays with mzpc5-specific primers. In situ hybridization analysis revealed that ovarian mzpc5 expression was restricted to the ooplasm of early (stage I-III) previtellogenic oocytes, and its expression signal weakened with oocyte growth. Following sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis with anti-mZPC5 antibodies, two immunoreactive proteins were detected in the ovary and chorion extracts. These proteins were approximately 50 and 74 kDa in size, like ZI-3 and ZI-2, respectively.Immunohistochemical assays using anti-mZPC5 and anti-Chg.H antibodies localized the mZPC5 protein in the ooplasm of early previtellogenic oocytes. With oocyte growth, mZPC5 tended to accumulate in the chorion, co-localizing with Chg.H.We previously showed that ovary-expressed ZP proteins could not compensate for Chg.L function loss in gene knock-out (chg.l -/-) medakas. As in our previous study, the chg.l-/- females produced oocytes with thin chorions, resulting in infertile soft eggs. However, in the present study, mZPC5 and Chg.H were co-localized in the chg.l-/- chorions. These results suggested that in the medaka previtellogenic oocyte, 1) mZPC5 is secreted from the ooplasm and deposited on the outer surface of its plasma membrane, creating the thin chorion layer; and 2) following the accumulation of liver-derived Chgs., the 3D structure of the chorion matrix is formed cooperatively with mZPC5 and Chgs. during oogenesis. More research is needed to confirm the functions of mZPC5 in chorion structure and physiology.

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression.

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Establishment of cytochrome P450 1a gene-knockout Javanese medaka, Oryzias javanicus, which distinguishes toxicity modes of the polycyclic aromatic hydrocarbons, pyrene and phenanthrene.

Cytochrome P450 1a (Cyp1a) is an important enzyme for metabolism of organic pollutants. To understand its reaction to polycyclic aromatic hydrocarbons (PAHs), we knocked out this gene in a marine model fish, Javanese medaka, Oryzias javanicus, using the CRISPR/Cas 9 system. A homozygous mutant (KO) strain with a four-base deletion was established using an environmental DNA (eDNA)-based genotyping technique. Subsequently, KO, heterozygous mutant (HT), and wild-type (WT) fish were exposed to model pollutants, pyrene and phenanthrene, and survivorship and swimming behavior were analyzed. Compared to WT, KO fish were more sensitive to pyrene, suggesting that Cyp1a transforms pyrene into less toxic metabolites. Conversely, WT fish were sensitive to phenanthrene, suggesting that metabolites transformed by Cyp1a are more toxic than the original compound. HT fish showed intermediate results. Thus, comparative use of KO and WT fish can distinguish modes of pollutant toxicity, providing a deeper understanding of fish catabolism of environmental pollutants.

Adenylate kinase 1 overexpression increases locomotor activity in medaka fish.

Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.

Targeted deletion of liver-expressed Choriogenin L results in the production of soft eggs and infertility in medaka, Oryzias latipes.

Egg envelopes (chorions) in medaka, Oryzias latipes, are composed of three major glycoproteins: ZI-1, - 2, and - 3. These gene-encoded chorion glycoproteins are expressed in the liver and/or ovarian oocytes of sexually mature female fish. In medaka, the glycoproteins produced in the female liver are induced by estrogen as Choriogenin (Chg.) H and Chg. H minor (m), which correspond to the zona pellucida (ZP) B (ZPB) protein in mammals, and Chg. L, which corresponds to ZPC in mammals. Chg. H, Chg. Hm, and Chg. L, are then converted to ZI-1, - 2, and - 3, respectively, during oogenesis in medaka ovaries.In the present study, we established a medaka line in which the chg.l gene was inactivated using the transcription activator-like effector nuclease (TALEN) technique. Neither intact chg.l transcripts nor Chg. L proteins were detected in livers of sexually mature female homozygotes for the mutation (homozygous chg.l knockout: chg.l-/-). The chg.l-/- females spawned string-like materials containing "smashed eggs." Closer examination revealed the oocytes in the ovaries of chg.l-/- females had thin chorions, particularly at the inner layer, despite a normal growth rate. In comparing chorions from normal (chg.l+/+) and chg.l-/- oocytes, the latter exhibited abnormal architecture in the chorion pore canals through which the oocyte microvilli pass. These microvilli mediate the nutritional exchange between the oocyte and surrounding spaces and promote sperm-egg interactions during fertilization. Thus, following in vitro fertilization, no embryos developed in the artificially inseminated oocytes isolated from chg.l-/- ovaries. These results demonstrated that medaka ZI-3 (Chg.L) is the major component of the inner layer of the chorion, as it supports and maintains the oocyte's structural shape, enabling it to withstand the pressures exerted against the chorion during spawning, and is essential for successful fertilization. Therefore, gene products of oocyte-specific ZP genes that may be expressed in medaka oocytes cannot compensate for the loss Chg. L function to produce offspring for this species.

The medaka fish Tol2 transposable element is in an early stage of decay: identification of a nonautonomous copy.

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations.

Establishment of culture and microinjection methods for false clownfish embryos without parental care.

Anemonefish, including the false clownfish Amphiprion ocellaris, are attractive model organisms because of their unique features, such as sex change and brilliant color patterns in mutants. However, anemonefish are not widely used to study gene function using reverse genetic approaches owing to microinjection difficulties and subsequent rearing and hatching of embryos without parental care. A. ocellaris embryos are spawned on a hard substrate and cared for by their parents until hatching. However, the eggs need to be detached from the substrate and raised without their parents to perform successful microinjection. We established a method to culture and hatch A. ocellaris embryos without spawning substrates or parental care. We found that changing water and generating water flow are critical for culturing the embryos, and that water flow (as physical stimulation) and complete darkness in the dark period are necessary for successful hatching. We further investigated the effectiveness of microinjection into the yolk sac of fertilized eggs rather than into the cytoplasm, which makes microinjection easier. A reporter RNA injected into the yolk sac was transferred to the cytoplasm and translated, indicating that yolk sac microinjection is an efficient alternative as has been used for zebrafish. These findings highlight the potential of A. ocellaris as an experimental model organism for reverse genetics, and our methods could be applied to other anemonefish species.

Deficiency of interleukin-17 receptor A1 induces microbiota disruption in the intestine of Japanese medaka, Oryzias latipes.

The mutual relationship between the intestinal immune system and the gut microbiota has received a great deal of attention. In mammals, interleukin-17A and F (IL-17A/F) are inflammatory cytokines and key regulators of the gut microbiota. However, in teleosts, the function of IL-17A/F in controlling the gut microbiota is poorly understood. We attempted to elucidate the importance of teleost IL-17 signaling in controlling gut microbiota. We previously established a knockout (KO) of IL-17 receptor A (RA) 1, a receptor for IL-17A/F, in the Japanese medaka (Oryzias latipes) using the CRISPR-Cas9 system and performed 16S rRNA-based metagenomic analyses using the anterior and posterior sections of the intestinal tract. The number of observed OTUs in the anterior intestine was significantly decreased in IL-17RA1 KO medaka compared to that in the wild-type (WT). Furthermore, ß-diversity analysis (weighted UniFrac) revealed considerably different bacterial composition in the anterior intestine of IL-17RA1 KO compared to WT, with similar findings in α-diversity. Notably, the pathogen Plesiomonas shigelloides was significantly increased in the posterior intestine of IL-17RA1 KO medaka. These findings indicate that signaling via IL-17RA1 is required to maintain a healthy gut microbiota in teleosts and mammals. The involvement of IL-17RA1 in controlling the gut microbiota has been demonstrated, resulting in microbiome dysbiosis in IL-17RA1 KO medaka.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

A defective interleukin-17 receptor A1 causes weight loss and intestinal metabolism-related gene downregulation in Japanese medaka, Oryzias latipes.

In the intestine, the host must be able to control the gut microbiota and efficiently absorb transiently supplied metabolites, at the risk of enormous infection. In mammals, the inflammatory cytokine interleukin (IL)-17A/F is one of the key mediators in the intestinal immune system. However, many functions of IL-17 in vertebrate intestines remain unclarified. In this study, we established a gene-knockout (KO) model of IL-17 receptor A1 (IL-17RA1, an IL-17A/F receptor) in Japanese medaka (Oryzias latipes) using genome editing technique, and the phenotypes were compared to wild type (WT) based on transcriptome analyses. Upon hatching, homozygous IL-17RA1-KO medaka mutants showed no significant morphological abnormality. However, after 4 months, significant weight decreases and reduced survival rates were observed in IL-17RA1-KO medaka. Comparison of gene-expression patterns in WT and IL-17RA1-KO medaka revealed that various metabolism- and immune-related genes were significantly down-regulated in IL-17RA1-KO medaka intestine, particularly genes related to mevalonate metabolism (mvda, acat2, hmgcs1, and hmgcra) and genes related to IL-17 signaling (such as il17c, il17a/f1, and rorc) were found to be decreased. Conversely, expression of genes related to cardiovascular system development, including fli1a, sox7, and notch1b in the anterior intestine, and that of genes related to oxidation-reduction processes including ugp2a, aoc1, and nos1 in posterior intestine was up-regulated in IL-17RA1-KO medaka. These findings show that IL-17RA regulated immune- and various metabolism-related genes in the intestine for maintaining the health of Japanese medaka.