Female Medaka Fish Research Articles

DIFFERENCES IN THE VULNERABILITY OF MEDAKA FISH (Oryzias celebensisis) MALE AND FEMALE TO CLORPYRIPHOS INSETICIDE

Chlorpyrifos is one of the most widely used organophosphate insecticides used to control pests on plants. The use of insecticides will produce residues in the soil and on plants and can be carried by rain flows to water bodies. This can pollute aquatic ecosystems and could negatively affected the growth of aquatic biota like Oryzias celebensis. This study aimed to determine the susceptibility of male and female medaka celebes (Oryzias celebensis) to chlorpyrifos insecticide. In this study, five fish medaka celebes were put in each jar in one jar for male medaka fish and female medaka fish with five replications. The parameters measured were survival rate, oxygen consumption rate between male and female of O. celebensis, temperature and pH. Statistically the survival rate of male and female medaka fish showed no statistically significant difference (P>0.05). Likewise, there was no difference between the oxygen consumption levels of male and female medaka fish before and after exposure to chlorpyrifos insecticide (P>0.05). This study concluded that there was no difference in susceptibility to chlorpyrifos insecticides between male and female medaka fish.

Toxicity, transfer and depuration of anatoxin-a (cyanobacterial neurotoxin) in medaka fish exposed by single-dose gavage

The proliferations of cyanobacteria are increasingly prevalent in many rivers and water bodies due especially to eutrophication. This work aims to study in female medaka fish the toxicity, the transfer and the depuration of the anatoxin-a, a neurotoxin produced by benthic cyanobacterial biofilms. This work will provide answers regarding acute toxicity induced by single gavage by anatoxin-a and to the risks of exposure by ingestion of contaminated fish flesh, considering that data on these aspects remain particularly limited.The oral LD50 and NOAEL of a single dose of (±)-anatoxin-a were determined at 11.50 and 6.67 μg.g−1, respectively. Subsequently, the toxico-kinetics of the (±)-anatoxin-a was observed in the guts, the livers and the muscles of female medaka fish for 10 days. Anatoxin-a was quantified by high-resolution qTOF mass spectrometry coupled upstream to a UHPLC chromatographic chain. The toxin could not be detected in the liver after 12 h, and in the gut and muscle after 3 days. Overall, the medaka fish do not appear to accumulate (±)-anatoxin-a and to largely recover after 24 h following a single sub-acute oral liquid exposure at the NOAEL.

Changes in the neurotransmitter profile in the central nervous system of marine medaka (Oryzias melastigma) after exposure to brevetoxin PbTx-1 – A multivariate approach to establish exposure biomarkers

A strategy to construct multivariate biomarkers for exposure to algal neurotoxins via correlating changes to the profiles of a series of neurotransmitters and their metabolites in the central nervous system (CNS) of exposed test organism is reported. 3-Month-old marine medaka (Oryzais melastigma) were exposed to waterborne brevetoxin PbTx-1 at two sub-lethal dose levels (0.5 and 2.5 μg-PbTx-1 L−1) for a duration of 12 h before quantification of 43 selected neurotransmitters and metabolites in their CNS were measured via dansyl chloride derivatization and LC-MS/MS determination. The profiling data were analyzed by multivariate statistical analyses, including principle component analysis (PCA), projection on latent structure-discriminate analysis (PLS-DA) and orthogonal projection on latent structure-discriminate analysis (OPLS-DA). Neurotransmitters and metabolites related to activation of voltage-gated sodium channels (VGSCs), N-methyl-D-aspartic acid receptors (NMDARs) and cholinergic neurotransmission were found to contribute significantly to class separation in the corresponding OPLS-DA models. Those models obtained from different exposure dosages were correlated by the Shared and Unique Structures Plot (SUS-plot) to identify appropriate variables for the construction of exposure biomarkers in the form of multivariate predictive scores. The established biomarkers for male and female medaka fish were able to predict acute sub-lethal exposure to PbTx-1 with good sensitivity and specificity (male fish: sensitivity 94.7%, specificity 80.0%; female fish: sensitivity 91.4%, specificity 83.3%). Neurotransmitter profiles in the CNS of medaka fish that should have recovered from exposure to PbTx-1 have also been determined to reveal long-term impacts to the CNS of the affected organism even after the exposure has been interrupted.

γ-H2AX foci as indication for the DNA damage in erythrocytes of medaka (Oryzias latipes) intoxicated with 4-nonylphenol

The present study aimed to investigate the genotoxicity in erythrocytes induced after exposure of medaka (Oryzias latipes) to 4-nonylphenol (4-NP). Adult female medaka fish were exposed to 4-NP at three sublethal concentrations for 15days to compare their sensitivity with that of catfish as an aquatic model. Comet assay and γ-H2AX were used as biomarkers to detect DNA damage in erythrocytes. Exposure to 4-NP resulted in an increase in the tail moment in a dose-dependent manner. The highest level of DNA damage was recorded after exposure to 100μg/l 4-NP. The number of foci was increased after exposure to 4-NP, indicating damage to DNA. The present results confirmed the high level of morphological alterations and apoptosis of erythrocytes detected in the first part of this study. 4-NP induced genotoxic effects in medaka, which were found to be more sensitive than catfish after exposure to 4-nonylphenol. Graphical abstract.

Immunostaining of UVA-induced DNA damage in erythrocytes of medaka (Oryzias latipes)

Some authors have recently reported that UVA induces double-strand breaks (DSBs) in DNA. Only a few researchers have reported on the induction of DSBs upon UVA exposure, as measured using the Comet assay and γ-H2AX as markers of DSB formation. In the present study, we have investigated for the first time the dose-dependent induction of DSBs by UVA in medaka (Oryzias latipes) erythrocytes. Adult female medaka fish were exposed to UVA for 15, 30, and 60min/day for three continuous days; an unirradiated control group was kept in the same laboratory conditions. At 0h and 24h after UVA exposure, blood was collected to detect DNA damage and repair. The number of γ-H2AX foci was higher than the control value at 0h after UVA exposure and decreased within a 24h. the comet assay showed that DNA repair began during the recovery period. These findings confirm our pervious findings of genotoxic effects after UVA exposure in medaka erythrocytes and suggest that the replication-independent formation of UVA-induced DSBs is mediated through the generation of reactive oxygen species. In conclusion, these results suggest that DNA damage and repair occur after UVA exposure in medaka fish. UVA is the main component of solar UV radiation and is used for artificial UV exposure. Our results may have implications for skin cancer research.

Hypoxia turns genotypic female medaka fish into phenotypic males.

Hypoxia caused by eutrophication is amongst the most pressing global problems in aquatic systems. Notably, more than 400 "dead zones" have been identified worldwide, resulting in large scale collapse of fisheries and major changes in the structure and trophodynamics. Recent studies further discovered that hypoxia can also disrupt sex hormone metabolism and alter the sexual differentiation of fish, resulting in male biased F1 generations and therefore posing a threat to the sustainability of natural populations. However, it is not known whether, and if so how, hypoxia can also change the sex ratio in vertebrates that have sex-determining XX/XY chromosomes. Using the Japanese medaka (Oryzias latipes) as a model, we demonstrate, for the first time, that hypoxia can turn genotypic female fish with XX chromosomes into phenotypic males. Over half of the XX females exposed to hypoxia exhibit male secondary sexual characteristics and develop testis instead of ovary. We further revealed that hypoxia can: (a) down-regulate the vasa gene, which controls proliferation of primordial germ cells and gonadal sex differentiation into ovary, and (b) up-regulate the DMY gene which resides at the sex-determining locus of the Y chromosome, and direct testis differentiation. This is the first report that hypoxia can directly act on genes that regulate sex determination and differentiation, thereby turning genotypic females into phenotypic males and leading to a male-dominant F1 population.

Modulation of Benzo[a]pyrene-Induced Toxic Effects in Japanese Medaka (Oryzias latipes) by 2,2′,4,4′-Tetrabromodiphenyl Ether

Because polycyclic aromatic hydrocarbons (PAHs) and polybrominated diphenylethers (PBDEs) are ubiquitous and coexist in the environment and in wildlife, there are potential interactions between them that could cause toxic effects. In this study, the modulating effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE47) and benzo[a]pyrene (BaP)-induced reproductive and developmental toxic effects in Japanese medaka (Oryzias latipes) were investigated by exposing adult Japanese medaka to BaP alone, BDE47 alone, and coexposing them with both BaP and BDE47 at different concentrations, respectively. Exposure to BaP alone significantly suppressed fecundity and egg protein content and markedly induced skeletal deformation in F1 generation eleutheroembryos. BDE47 significantly recovered reproductive functions, fecundity, and egg protein content, suppressed by BaP when the concentration of BDE47 increased to 0.44 μg/L. Such effects can be at least partly explained by the decreased BaP levels in the coexposure groups and the accompanying increase in the circulating level of 17β-estradiol in female medaka fish. The prevalence of skeletal deformations markedly increased to 19.3 ± 2.4% and 16.0 ± 1.6% in fish coexposed to BaP and BDE47 at 0.44 and 2.58 μg/L compared with that of fish exposed to 1.21 μg/L BaP alone (9.7 ± 1.7%), and the impacts on male medaka fish in the coexposure groups would be the crucial reason leading to these effects. Considering that the measured water concentrations of BaP and BDE47 in the present study were comparable with those reported in rivers and harbors, BaP and BDE47 contamination in the real world would have a significant level of interactive effects on wild fish.

Generation of Medaka Fish Haploid Embryonic Stem Cells

Haploid embryonic stem (ES) cells combine haploidy and pluripotency, enabling direct genetic analyses of recessive phenotypes in vertebrate cells. Haploid cells have been elusive for culture, due to their inferior growth and genomic instability. Here, we generated gynogenetic medaka embryos and obtained three haploid ES cell lines that retained pluripotency and competitive growth. Upon nuclear transfer into unfertilized oocytes, the haploid ES cells, even after genetic engineering, generated viable offspring capable of germline transmission. Hence, haploid medaka ES cells stably maintain normal growth, pluripotency, and genomic integrity. Mosaic oocytes created by combining a mitotic nucleus and a meiotic nucleus can generate fertile fish offspring. Haploid ES cells may offer a yeast-like system for analyzing recessive phenotypes in numerous cell lineages of vertebrates in vitro.

Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals

There are multiple populations of gonadotropin-releasing hormone (GnRH) neurons in the brains of vertebrates. The population located in the hypothalamus/preoptic area is the best studied and is known to ultimately control reproduction. Teleost fish have an additional population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulbs, the physiological function of which is still unclear. Anatomical and physiological studies provide evidence that TN-GnRH neurons have extensive projections in the brain and modulate neuronal activity. Although there is anatomical evidence that the TN receives olfactory and optic sensory inputs, it is not known if sensory information is transmitted to TN-GnRH neurons to modulate their activity. In the present study, we tested the hypothesis that social cues from conspecifics modulate electrical activity of TN-GnRH neurons from the intact brain of female medaka fish (Oryzias latipes). We further investigated the potential roles of chemosensory and visual signals in mediating the social cue response. We used a transgenic line of medaka with TN-GnRH neurons genetically tagged with green fluorescent protein, allowing visualization of specific neurons for whole-cell current clamp electrophysiology. We demonstrated that 24-h exposure to male visual and chemosensory cues suppressed the electrical activity of female TN-GnRH neurons compared with exposure to other females. Chemosensory cues alone were insufficient to induce this social cue response. However, visual cues alone replicated the "combined" social cue response. These findings support our hypothesis that sensory signals--and specifically, visual social cues--modulate electrical activity of TN-GnRH neurons.

Comparative Susceptibility of Kidney and Gill Cells of Medaka Fish (Oryzias latipes) to Spontaneous and X-ray-induced Micronucleus Formation

The frequency of micronucleated cells (MNCs) was measured in acridine orange (AO) stained kidney cells obtained from male and female medaka fish (Oryzias latipes) of known body weight before and after X-irradiation at a dose of 4 Gy. The MNC frequency in the kidney cells obtained from untreated and X-ray treated fish did not vary in correlation with the body weight in either gender, and neither did it show a significant gender-related difference. In both the untreated and treated groups, the number of MNCs in kidney cells per fish appeared to follow a Poisson distribution. These results are in agreement with those previously reported for the MNC frequency in gill cells of medaka fish. The average MNC frequency in kidney cells for the untreated fish was 0.4‰ (12/30000), which was significantly lower than 0.8‰ (24/30000) reported for gill cells. Similarly, a relatively lower frequency of X-ray-induced MNCs was evident for kidney cells (2.2‰; 69/30000) vs. gill cells (6.3‰; 191/30000). These results combined with previous reports support the use of AO to detect micronucleus formation in kidney cells, and further indicate that the kidney cell micronucleus assay can be used as a reliable and coincidently applied adjunct to the gill cell assay for medaka fish.

Vasotocin/isotocin-immunoreactive neurons are decreased after spwaning in the female medaka fish ( Oryzias latipes) brain

Vasotocin/isotocin-immunoreactive neurons are decreased after spwaning in the female medaka fish ( Oryzias latipes) brain

Susceptibility of male and female medaka ( Oryzias latipes) fish to spontaneous and X-ray induced micronucleus formation in gill cells

The frequency of micronucleated cells (MNCs) was measured in acridine-orange (AO) stained RNA-rich gill cells from male and female medaka ( Oryzias latipes) fish of known body weight. Spontaneous MNC frequencies were not significantly correlated with body weight, despite the fact that the heaviest of the 30 fish used outweighed the lightest by a factor of 3. Average MNC frequencies were identical in males and females at 0.8‰. An X-ray dose of 4 Gy increased the frequency of MNCs over the spontaneous level in all 30 of the fish used, reaching a level of 7.2‰ on average when assayed 24 h after exposure. In X-ray treated fish, MNC frequency and body weight were not significantly correlated, nor was there any difference between the sexes. These and other results support our primary conclusion that AO-staining is suitable for the medaka micronucleus assay in gill cells, and indicate that male and female medaka fish are similarly and size-independently susceptible to both spontaneous and X-ray induced micronucleus formation in gill cells.