Anesthetic effects of clove oil and lidocaine-HCl on marine medaka (Oryzias dancena)

Fish may be anesthetized for various experimental and practical purposes, primarily to immobilize them in order to facilitate handling. Marine medaka (Oryzias dancena) is a teleost fish used in marine ecotoxicology studies. Despite the importance of anesthesia in handling experimental fish, the effects of anesthesia in marine medaka have not yet been investigated. In this study, the authors evaluated the anesthetic effects (time required for anesthesia to take effect and recovery time) of two anesthetic agents, clove oil and lidocaine-HCl, on marine medaka. They anesthetized fish at different water temperatures (23 °C, 26 °C and 29 °C) and using different concentrations of clove oil (50 ppm, 75 ppm, 100 ppm, 125 ppm, 150 ppm and 175 ppm) or lidocaine-HCl (300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm and 800 ppm). The time required for anesthesia to take effect decreased significantly as both anesthetic concentration and water temperature increased for both clove oil and lidocaine-HCl. To anesthetize marine medaka within approximately 1 min, the optimal concentrations for clove oil were 125 ppm at 23 °C, 100 ppm at 26 °C and 75 ppm at 29 °C and for lidocaine-HCl were 800 ppm at 23 °C and 700 ppm at both 26 °C and 29 °C. The authors also compared anesthetic effects in marine medaka of different sizes. Both anesthetic exposure time and recovery time were significantly shorter for smaller fish than for larger fish. These results provide a useful foundation for the laboratory handling of marine medaka.     

Temperature-dependent growth rates and gene expression patterns of various medaka Oryzias latipes cell lines derived from different populations

Medaka Oryzias latipes has several geographically and genetically distinct populations. We examined temperature acclimation response in various medaka cell lines derived from different populations. Measurement of cell growth at various temperatures suggested that 15°C was the permissive growth temperature in all cell lines from the Northern Japanese and East Korean populations, but not in those from the Southern Japanese population and medaka-related species Oryzias celebensis, which inhabits a tropical zone. RT-PCR for 102 temperature-responsive genes, previously reported in other species, revealed that the accumulated mRNA level of a gene encoding HSP47 was lower at 25°C than at 33°C, and vice versa for 12 genes including IκBα and Rab-1c, in OLHNI-1 cell line from the Northern Japanese population. Further analysis by real-time PCR demonstrated that the accumulated mRNA levels of IκBα and Rab-1c in OLHNI-1 and OLSOK-e7 cell lines from the East Korean population were increased when the culture temperature was shifted from 33 to 15°C, but not in OLHdrR-e3 cell line from the Southern Japanese population. Since IκBα and Rab-1c are related to the NFκB cascade and endoplasmic reticulum-to-Golgi transport, respectively, it is inferred that immune responses and intracellular transport are possibly critical to temperature adaptation for medaka. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes)

In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.     

Complete mitochondrial genome of the marine medaka Oryzias melastigma (Beloniformes, Adrianichthyidae)

The complete mitochondrial genome was obtained from the assembled genome data sequenced by next-generation sequencer from the marine medaka Oryzias melastigma. The mitochondrial genome sequence was 16,864 bp in size, and the gene order and contents were identical with those of previously reported fish mitochondrial genomes. Of 13 protein-coding genes (PCGs), 4 genes (CO3, ND3, ND4, and Cytb) had incomplete stop codons. The base composition of O. melastigma mitogenome showed high A+T (59.65%) and anti-G bias (8.73%) on the 3rd position of PCGs.     

Characterization of estrogen-responsive transgenic marine medaka Oryzias dancena germlines harboring red fluorescent protein gene under the control by endogenous choriogenin H promoter

Transgenic marine medaka (Oryzias dancena) germlines were generated by the microinjection of the red fluorescent protein (RFP) reporter gene (rfp) driven by the endogenous choriogenin H gene (chgH) promoter. The selected transgenic lines contained multiple copies of the transgene (3–42 copies per cell) in their genomes. Although all the founders were mosaic, the transgene was stably transmitted from the F1 generation to all subsequent generations following a Mendelian pattern. Different transgenic lines showed different responsiveness to estradiol-17β (E2) exposure at the mRNA and protein levels, and the expression efficiency was dependent upon the transgene copy number. The induction of RFP was significantly affected by the developmental stage of transgenic larvae: later-stage larvae (older than 7 days post-hatching) showed higher sensitivity to E2 exposure than earlier-stage larvae. The response of transgenic expression to E2 was fairly dependent upon the E2 dose (200–3,200 ng/L) and exposure period (1–7 days), according to both a microscopic examination of RFP intensity and a qRT–PCR assay. The transgenic marine medaka showed similar transgenic responses to E2 under freshwater, brackish, and seawater conditions. In addition to E2, the transgenic RFP signal was also successfully induced during 1-week exposure to various other natural (1 μg/L estrone and 10 μg/L estriol) and synthetic (xeno)estrogens (0.1 μg/L 17α-ethynylestradiol, 1 μg/L diethylstilbestrol, and 10 mg/L bisphenol A). The efficiency of transgene expression varied greatly among the chemicals tested. The results of this study suggest that the chgH–rfp transgenic marine medaka species will be useful in the in vivo detection of waterborne estrogens under a wide range of salinity conditions.     

The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments

The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl^? through the transporters including the Na⁺, K⁺-ATPase (NKA), Na⁺, K⁺, 2Cl^? cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl^? secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (> 1 day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24 h, the Cl^? secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments.     

Fish cell culture: Establishment of two fibroblast-like cell lines (OL-17 and OL-32) from fins of the medaka,Oryzias latipes

Summary Two fibroblast-like cell lines were obtained from fins of adults of the medaka,Oryzias latipes, and serially cultured at 27° C. One cell line, which was derived from a fish of the orange-red variety, reached to population doubling level (PDL) 434 on Day 840 by 120 passages. The other, which was derived from a fish of the inbred strain HB32C, reached to PDL 294 on Day 551 by 80 passages. Any symptom of crisis was not detected. The cell lines were named OL-17 and OL-32, respectively. Population doubling time was 29 h for OL-17 cells, and 32 h for OL-32 cells. Density-dependent inhibition of growth was clear in OL-32 cells, but not so obvious in OL-17 cells. Modal chromosome number of OL-17 cells was 50, and that of OL-32 cells was 47 (2N=48). Plating efficiency of OL-17 cells was about 10%, whereas that of OL-32 cells was about 5%. The use of conditioned medium (80% concentration) increased the plating efficiency of OL-32 cells to more than 25%.     

Comparative study of embryonic thermoresistance of two inbred strains of the medaka (Oryzias latipes)

Summary By using inbred strains (HO4C and HB32C) of the medaka,Oryzias latipes, the involvement of genetic factor(s) in the determination of thermoresistance of fish was investigated. The thermoresistance of embryos of the medaka was quantitated by the fraction of the embryos surviving 1 day after heat treatment. At early stages of development (st. 13 and st. 20–21), the HO4C strain was more resistant than the HB32C strain. At st. 20–21, the HO4C strain was more resistant than the HB32C strain at all temperatures used (42, 43, and 44°C). At later stages of development (st. 27 and st. 32), however, the HB32C strain was more resistant than the HO4C strain.The results of genetic cross experiments raised the following possibilities; the thermoresistance of embryos at early developmental stages can be lowered by some factor(s) inherited in the HO4C strain and/or increased by those in the HB32C strain. By contrast, the sensitivity of embryos at later stages of development was not affected by factor(s) of their parents, but by their own genetic constitution.     

Characterization and expression of embryonic and adult globins of the teleost Oryzias latipes (medaka)

Using the teleost Oryzias latipes (medaka), we isolated three embryonic globin cDNAs (em.alpha-0, em.alpha-1, and em.beta-1) from the embryos 5 days after fertilization (at 30 degrees C) and two adult globin cDNAs (ad.alpha-1 and ad.beta-1) from the kidney of the fully-grown adult fish, and predicted their amino acid sequences. Molecular phylogenetic analysis showed that the embryonic globins were highly homologous in amino acid sequence to the embryonic globins previously identified in rainbow trout and zebrafish, and that they formed a monophyletic group among the teleostean globin molecules. They were clearly discriminated from the adult globin of the medaka. RT-PCR analysis showed that the embryonic globin mRNAs were intensely expressed in stage 30 and 38 embryos and in young fish 30 days after hatching. The level of expression decreased drastically after the young fish stage, and was low in fully-grown adult fish. The adult alpha globin mRNA ad.alpha-1 was scarcely expressed in the embryos, and the level of expression gradually increased in young to fully-grown adult fish. Unexpectedly, the adult beta globin mRNA ad.beta-1 was expressed throughout life, from the early embryonic stage to the fully-grown adult stage. This expression profile was quite different from that of the rainbow trout previously investigated. Some globins of the medaka were expressed both in primitive hematopoiesis and in definitive hematopoiesis.     

Human embryonic stem cell lines derived from the Chinese population

Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.     

Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

Summary Species of small fish are becoming useful tools for studies on vertebrate development. We have investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporal and spatial expression patterns of bacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as well as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.This work contains part of the PhD thesis of C. Winkler     

Identification of robust hypoxia biomarker candidates from fin of medaka (Oryzias latipes)

Aquatic hypoxia caused by organic pollution and eutrophication is a pressing worldwide water pollution problem. Better methods for monitoring oxygen levels are needed to assist efforts to maintain and protect the health of natural aquatic environments. In this project, we used a Japanese ricefish (medaka, Oryzias latipes) 8K oligonucleotide array as a platform to identify potential hypoxic biomarkers in different organs (fin, gill, liver and brain) upon exposure to hypoxia. The microarray results were validated by qRT-PCR employing a subset of candidate biomarkers. Interestingly, the largest number and most significant of hypoxia responding array features were detected in hypoxia exposed fin tissues. We identified 173 array features that exhibited a significant response (over 2 fold change in expression) upon exposure to hypoxic conditions and validated a subset of these by quantitative RT-PCR. These gene targets were subjected to annotation and gene ontology mining. Positively identifiable gene targets that may be useful for development of a rapid and accurate biomarker test using fin clips are discussed in relation to previous reports on hypoxia responsive genes.     

Specification of primordial germ cells in medaka (Oryzias latipes)

Background  

Primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types.