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The MRE11A-RAD50-NBS1 complex activates the ataxia-telangiectasia mutated (ATM) pathway and plays a central role in genome homeostasis. The association of RAD50 mutations with disease remains unclear; hence, we adopted a medaka rad50 mutant to demonstrate the significance of RAD50 mutation in pathogenesis using the medaka as an experimental animal. A 2-base pair deletion in the rad50 gene was introduced into transparent STIII medaka using the CRISPR/Cas9 system. The mutant was analyzed histologically for tumorigenicity and hindbrain quality, as well as for swimming behavior, to compare with existing ATM-, MRE11A-, and NBS1-mutation-related pathology. Our results revealed that the medaka rad50 mutation concurrently reproduced tumorigenesis (8 out of 10 rad50Δ2/+ medaka), had a decrease in median survival time (65.7 ± 1.1 weeks in control vs. 54.2 ± 2.6 weeks in rad50Δ2/+ medaka, p = 0.001, Welch's t-test), exhibited semi-lethality in rad50Δ2/Δ2 medaka and most of the major ataxia-telangiectasia phenotypes, including ataxia (rheotaxis ability was lower in rad50Δ2/+ medaka than in the control, Mann-Whitney U test, p < 0.05), and telangiectasia (6 out of 10 rad50Δ2/+ medaka). The fish model may aid in further understanding the tumorigenesis and phenotype of ataxia-telangiectasia-related RAD50 germline mutations and in developing novel therapeutic strategies against RAD50 molecular disorders., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chisada et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Many studies using experimental and wild animals have reported negative effects of microplastic beads (MPs) ingestion. However, data regarding the lowest observed adverse effect levels (LOAELs) of MPs remain limited. Our aim was to evaluate the adverse effect levels of polyethylene MPs (10-63 μm diameter) with respect to growth, reproduction, and the eyes and kidneys of medaka (Oryzias latipes) under breeding conditions to contribute to future research involving LOAEL determinations. Fish were exposed to 0.009 mg-MPs (approximately 1000 particles)/L to 0.32 mg-MPs (approximately 40,000 particles)/L for 12 weeks. The eyes and kidneys were evaluated by histopathologic analysis. Although histologic analyses indicated an absence of MPs in the tissues, the eyes and kidneys as well as reproduction were adversely affected by increasing MP concentrations. The number of spawned eggs decreased, and changes were noted in the eyes of fish exposed to ≥0.032 mg-MPs/L under breeding conditions. The eyes exhibited thinning of the optic nerve fiber layer and dilatation of retinal capillaries compared with medaka not treated with MPs. Changes in the kidneys were observed in fish exposed to ≥0.065 mg-MPs/L. The mesangial matrix in the glomerulus of the kidneys was expanded compared with non-treated medaka, suggesting a deterioration in renal function. Analyses of an oxidative stress marker in the tissues indicated that lesion progression was associated with increased oxidative stress. Furthermore, a comparison of adverse effect levels suggested that MPs were more toxic to the eyes and reproduction than the kidneys or growth. Our data should prove useful for determining the LOAELs of polyethylene beads on vertebrates and enhance understanding of the mechanism underlying the biological toxicity of polyethylene MPs., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Research using various species of wild and cultured fish has identified negative effects of short-term exposure to microbeads. Although wild animals might be contaminated with microbeads and/or other pharmaceuticals, data regarding the long-term effects remain limited. To clearly elucidate the effects of microbeads, studies of long-term exposure using animal models are necessary. Our aim was to elucidate the effects of microbeads alone on the growth and fecundity of medaka following long-term exposure (12 weeks). In experiment 1, fish groups (except controls) were temporarily exposed to polyethylene microbeads (10-63 μm diameter) a low dose of 0.065 microbeads-mg/L and high dose of 0.65 microbeads-mg/L. In experiment 2, see-through medaka and fluorescent polyethylene microbeads (10-45 μm diameter) were used to estimate the retention time of ingested microbeads in the digestive tract, which was 4-9 days. The low dose of microbeads did not affect growth but did decrease the number of eggs and the hatching rate. The high dose decreased growth, the number of eggs, and hatching rate. Growth differences were recognized for the first time at 7 weeks, and differences in the number of eggs at 12 weeks. Thus, long-term tests using medaka indicated that microbeads per se exhibit growth inhibition and reproductive toxicity. These effects could be associated with nutritional factors resulting from the long retention time of microbeads in the digestive tract. We also determined the dose that affects only fecundity. This suggests that normal growth of medaka in the wild does not mean the environment is free from microbead contamination. We are thus attempting to identify new biological indexes for monitoring the status of microbead contamination using our system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this study was to elucidate the renal lesions of leptin receptor-deficient medaka showing hyperglycemia and hypoinsulinemia and to evaluate the usefulness of the medaka as a model of diabetic nephropathy. Leptin receptor-deficient medaka at 20 and 30 weeks of age showed hyperglycemia and hypoinsulinemia; they also showed a higher level of plasma creatinine than the control medaka. Histopathologically, dilation of glomerular capillary lumina and of afferent/efferent arterioles was observed in leptin receptor-deficient medaka at 20 weeks of age, and then glomerular enlargement with cell proliferation and matrix expansion, formation of fibrin cap-like lesions, glomerular atrophy with Bowman's capsule dilation, and renal tubule dilation were observed at 30 weeks of age. These histopathological characteristics of leptin receptor-deficient medaka were similar to the characteristics of kidney lesions of human and rodent models of type II diabetes mellitus, making leptin receptor-deficient medaka a useful model of diabetic nephropathy., (©2019 The Japanese Society of Toxicologic Pathology.)

Background: In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported., Results: In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries., Conclusions: These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

We have previously reported that fish pathogens causing vibriosis specifically adhere to GM4 on the epithelial cells of fish intestinal tracts (Chisada, S., Horibata, Y., Hama, Y., Inagaki, M., Furuya, N., Okino, N., and Ito, M. (2005) Biochem. Biophys. Res. Commun. 333, 367-373). To identify the gene encoding the enzyme for GM4 synthesis in the fish intestinal tract, a phylogenetic tree of vertebrate ST3GalVs, including Danio rerio and Oryzias latipes, was generated in which two putative subfamilies of fish ST3GalVs were found. Two putative ST3GalVs of zebrafish (zST3GalV-1 and -2), each belonging to different subfamilies, were cloned from the zebrafish cDNA library. Interestingly, zST3GalV-1 synthesized GM3 (NeuAcalpha2-3Galbeta1-4Glcbeta1-1'Cer) but not GM4, whereas zSTGalV-2 synthesized both gangliosides in vitro when expressed in CHO-K1 and RPMI1846 cells. Flow cytometric analysis using anti-GM4 antibody revealed that the transformation of RPMI1846 cells with zST3GalV-2 but not zST3GalV-1 cDNA increased the cell-surface expression of GM4. Whole mount in situ hybridization showed that the zST3GalV-2 transcript was strongly expressed in the gastrointestinal tract, whereas zST3GalV-1 was expressed in the brain and esophagus but not gastrointestinal tract in 3-day post-fertilization embryos. It has long been a matter of controversy which enzyme is responsible for the synthesis of GM4 in mammals. We found that three isoforms of mouse ST3GalV (mST3GalV) having different N-terminal sequences can synthesize GM4 as well as GM3 when expressed in RPMI1846 and CHO-K1 cells. Furthermore, mST3GalV knock-out mice were found to lack GM4 synthase activity and GM4 in contrast to wild-type mice. These results clearly indicate that zST3GalV-2 and mST3GalV are the enzymes responsible for the synthesis of GM4 in zebrafish and mice, respectively.