Hermaphroditic Fish Rivulus Marmoratus Research Articles

Molecular cloning and characterization of θ-class glutathione S-transferase (GST-T) from the hermaphroditic fish Rivulus marmoratus and biochemical comparisons with α-class glutathione S-transferase (GST-A)

We cloned and sequenced full-length cDNA of a θ-class-like glutathione S-transferase (GST-T) from liver tissue of the self-fertilizing fish Rivulus marmoratus. The full-length cDNA of rm-GST-T was 907 bp in length containing an open reading frame of 666 bp that encoded a 221-amino acid putative protein. Its derived amino acid sequence was clustered with other vertebrate θ-class GSTs in a phylogenetic tree. The deduced amino acid sequence of θ-like rm-GST (rm-GST-T) was compared with both classes (α and θ) of GST and α-class rm-GST (rm-GST-A). Tissue-specific expression of two rm-GST mRNAs was investigated using real-time RT-PCR. To further characterize the catalytic properties of this enzyme along with rm-GST-A, we constructed the recombinant θ-like rm-GST plasmid with a 6×His-Tag at the N-terminal of rm-GST-T cDNA. Recombinant rm-GST-T was highly expressed in transformed Escherichia coli, and its soluble fraction was purified by His-Tag affinity column chromatography. The kinetic properties and effects of pH and temperature on rm-GST-T were further studied, along with enzyme activity and inhibition effects, and compared with recombinant rm-GST-A. These results suggest that recombinant rm-GSTs such as rm-GST-A and rm-GST-T play a conserved functional role in R. marmoratus.

Nonylphenol modulates expression of androgen receptor and estrogen receptor genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERα and ERβ) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERα, and ERβ from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERα, and ERβ genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ER α and Rm-ERβ cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERβ mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERα mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERα mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERβ mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 μg/l) and BisA (600 μg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERα, and Rm-ERβ mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERα, and Rm-ERβ genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.

Endocrine disrupting chemicals (bisphenol A, 4-nonylphenol, 4-tert-octylphenol) modulate expression of two distinct cytochrome P450 aromatase genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To understand the effect of endocrine-disrupting chemicals (EDCs) on cytochrome P450 aromatase ( rm- cyp19) gene expression between gender types in the hermaphroditic fish Rivulus marmoratus, we cloned two distinct rm- cyp19 genes using RT-PCR with degenerative primers, obtained full-length cDNAs using 5′- and 3′-RACE-PCR methods, and completely sequenced them. The brain aromatase ( rm- cyp19b) cDNA consisted of 2,124 bp including the open reading frame (ORF), which encoded a putative protein of 505 amino acids. The ovarian aromatase ( rm-cyp19a) cDNA consisted of 2,075 bp, including the ORF encoding a putative protein of 516 amino acids. Expression patterns of rm- cyp19b and rm-cyp19a mRNAs were investigated in embryos of different developmental stages and in seven different tissues of adult fish. The rm- cyp19b gene in hermaphrodite and secondary male R. marmoratus was predominantly expressed in the brain, while the rm- cyp19a gene was expressed gender-specifically in the gonad. The expression of rm- cyp19b mRNA increased from stage 1 (2d post fertilization) to stage 4 (12d post fertilization) in a developmental stage-dependent manner but steeply decreased in the hatching stage. Compared to the rm- cyp19b gene, the abundance of ovarian aromatase rm- cyp19a transcripts was very low, and its expression was first detected at stage 3 and then decreased gradually to the hatching stage. Alteration of rm- cyp19b and rm- cyp19a gene expression was further analyzed in the brain and gonad by real-time RT-PCR 96 h after EDC exposure in hermaphrodites and secondary males. The brain aromatase rm- cyp19b gene was up-regulated in the brain after 4-nonylphenol (4-NP)-exposure, while the ovarian aromatase rm- cyp19a gene was significantly down-regulated in the gonad. In 300 μg/L 4-tert octylphenol (4-tert-OP), or 600 μg/L bisphenol A-exposed brain and gonad, both rm- cyp19b and rm- cyp19a genes were up-regulated. In the case of secondary males, the rm- cyp19b gene was highly expressed in the 4-NP-exposed brain, while expression of the rm- cyp19a gene was not detected in the gonad. These results indicate that the expression of rm- cyp19a and rm- cyp19b genes is differently modulated according to estrogenic compounds and gender type of R. marmoratus.

Cloning of Ki-ras and Ha-ras cDNAs from the hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and its expression after exposure to 4-nonylphenol

Previous studies on ras proto-oncogene genes in fish have been focused on chemical-associated carcinogenesis, and the expression of fish ras genes was not well-characterized. We investigated Ki- and Ha-ras genes from the hermaphroditic fish Rivulus marmoratus to understand better their expression patterns in specific tissues, as well as their responses to endocrine-disrupting chemicals such as 4-nonylphenol (4-NP). By investigating expression patterns, we found that the R. marmoratus Ki-ras (Rm Ki-ras) gene showed an alternative splicing event between exons 4A and 4B according to tissue types, which is different from the expression pattern of mammalian Ki-ras genes. In the Rm Ki-ras gene, there were two different expressed types, with exons 1-2-3-4A-4B (long form) and with exons 1-2-3-4B (short form). In the Rm Ki-ras gene, the long form was expressed strongly in the gonad and intestine, and the short form was expressed ubiquitously, except for a low level of expression in the liver. Following 4-NP exposure (300 microg/L), the Rm Ki-ras long form in the liver was significantly expressed, while it was expressed moderately in the ovaries. However, the Rm Ha-ras gene was significantly over-expressed in the brain, while its expression in the gonad was down-regulated. In relation to these modulations after 4-NP exposure, we searched the Rm Ha- and Ki-ras promoter regions and found several ERE-half sites, that may be involved in the modulation of ras gene expression following 4-NP exposure. These genes could be applicable as new biomarker genes for assessing exposure to endocrine-disrupting chemicals (EDCs). Further, this implies the disturbance of ras-dependent signal transduction following EDC exposure.

CDNA cloning and expression of a cytochrome P450 1A ( CYP1A) gene from the hermaphroditic fish Rivulus marmoratus

We previously described the genomic structure of the cytochrome P450 1A ( CYP1A) gene from the hermaphroditic fish Rivulus marmoratus [Kim, I.-C., Kim, Y.J., Yoon, Y.-D, Kawamura, S., Lee, Y.-S., Lee, J.-S., 2004a. Cloning of cytochrome P450 1A ( CYP1A) genes from the hermaphroditic fish Rivulus marmoratus and the Japanese medaka Oryzias latipes. Mar. Environ. Res. 58, 125–129]. To further characterize R. marmoratus CYP1A, we cloned the cDNA sequence of a CYP1A gene from this species and also expressed its recombinant protein in an E. coli system. We exposed R. marmoratus to 4-nonylphenol, and found a small induction of CYP1A mRNA in the treated animals. In this paper, we discuss the characteristics of R. marmoratus CYP1A gene as well as its potential use in a biomonitoring assay.

Cloning and expression of alpha class glutathione S-transferase gene from the small hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

In order to assess its potential as a biomarker of aquatic pollution, an alpha class glutathione S-transferase gene (GSTα gene) was cloned from the small hermaphroditic fish Rivulus marmoratus. The R. marmoratus GSTα gene spanned 1.3 kb, consisting of 6 exons encoding 221 amino acid residues. It showed high similarity to zebrafish GST. We named this R. marmoratus GSTα gene as rm-GSTα. The cDNA of the rm-GSTα gene was also investigated for its phylogeny, tissue-specific and chemical-induced expression. Rm-GSTα was subcloned into a 6 × His-tagged pCR ®T7 TOPO TA expression vector to produce the recombinant 6 × His-tagged rm-GST protein. This will be used in future to raise an rm-GSTα antibody for use in the study of phase II metabolism involved in detoxification. We also exposed R. marmoratus to 300 μg/l of 4-nonylphenol in water, and found approximately 4-fold induction of R. marmoratus GSTα mRNA in the treated animals. In this paper, we discuss the characteristics of the R. marmoratus GSTα gene as well as its potential use in relation to environmental pollution.

Effects of Acute Acid Stress on Hatching and Mortality of Hermaphroditic Teleost, Rivulus marmoratus (Cyprinodontiformes; Rivulidae)

The effects of acute acid stress on hatching success and hatching period of laboratory‐reared hermaphroditic fish Rivulus marmoratus were examined. The effects of acute acid toxicity on mortality was also determined in three life stages of this fish. There was a significant negative effect of acid stress on hatching performance in the R. marmoratus embryos. The hatching success was only 5% at pH 3.5 compared to over 78% at pH higher than 4.0. The hatching period was also delayed by low pH treatments. The larval and juvenile stages were more sensitive to acid toxicity on mortality than the adult stage, but larvae and juveniles showed similar sensitivity. The 96‐h LC50 value was pH 3.8 in larval and juvenile stages and pH 3.3 in adult stage.

Age, Growth, and Sexual Development in the Self-fertilizing Hermaphroditic Fish Rivulus marmoratus

We studied age, growth, and sexual development in the early life intervals of the self-fertilizing mangrove killi-fish, Rivulus marmoratus. Newly hatched (day 0) individuals had sagittal otoliths of 60 μm radius, with about 30 increments. Sequential sampling until about day 60 after hatching yielded otoliths with the number of increments outside the 60 μm radius equal to the daily age of the fish. Alizarin complexone marking of otoliths also confirmed the increments were daily, and demonstrated the applicability of this technique to field studies for capture-mark-recapture, or age and growth estimates. Individuals fed a restricted amount of food formed fewer daily otolith growth increments than fish fed to satiation each day. Using histological analysis for identifying gonad morphogenesis, we found no correlation between gonadal development and external appearance (caudal ocellus, orange fin colouration) in young fish of known ages. The caudal ocellus was not present until 9 mm total length, and developed thereafter. Of 136 individuals examined, fish less than 17.2 mm total length (TL, n = 124) were females. Testicular tissue first appeared among individuals 17–18 mm TL (n = 3), while some individuals greater than 18 mm TL (n = 8) were functional hermaphrodites. The single male in our study was relatively of small body size (9.6 mm, day 37) with a distinct caudal ocellus, indicating that it is presumably a primary male.

The Internally Self-fertilizing Hermaphroditic Teleost Rivulus marmoratus (Cyprinodontiformes, Rivulidae) β-Actin Gene: Amplification and Sequence Analysis with Conserved Primers

To determine the ease and feasibility of amplifying the beta-actin gene in fish by the polymerase chain reaction (PCR), genomic DNAs of several fish (Rivulus, Southern top mouth minnow, common fat minnow, oily bitterling, carp, Far Eastern catfish, medaka, and European flounder) were extracted and used as a template with conserved primers, designed on the basis of high amino acid homology (approximately 98% or more). Among them, the self-fertilizing hermaphroditic fish Rivulus marmoratus was chosen for further characterization. After amplification of the Rivulus beta-actin PCR product with Taq polymerase, PCR product was subcloned to pCRII vector. After restriction enzyme mapping of Rivulus beta-actin gene, the amplified insert was sequenced using ALF Express automatic DNA sequencer with conserved internal primers. The R. marmoratus beta-actin gene consists of 1763 bp encoding 375 amino acids including 5 exons and 4 introns. The splicing and acceptance sites of the exon and intron boundaries of the Rivulus beta-actin gene were highly conserved with consensus sequences (GT/AG). The amino acid homology of R. marmoratus beta-actin to other species was high: 98.93% to human; 98.93%, Atlantic salmon; 98.93%, common carp; 98.93%, grass carp; 98.93%, zebrafish; 98.67%, medaka; and 98.40%, sea bream. To determine the expression of the R. marmoratus beta-actin gene in liver and ovary, reverse transcriptase-polymerase chain reaction was carried out with internal primers. In conclusion, these universal primers are successful in the rapid cloning of the fish beta-actin gene by PCR, based on a high homology of the beta-actin gene conserved through evolution. This approach will be applicable to the isolation of other beta-actin homologues in the investigation of phylogenetic comparisons of fish species, along with a possible application to cloning strategy in other conserved genes.

Nmethyl-N-nitrosourea (MNU) induces papillary thyroid tumours which lack ras gene mutations in the hermaphroditic fish Rivulus marmoratus.

To investigate the patterns of alkylating agent-induced tumour formation, 40 fish (Rivulus marmoratus) were exposed to N-methyl-N-nitrosourea (MNU) at 50 ppm in 10 mM Hepes-buffered synthetic seawater for 2 h. Tumour incidence 4 months after exposure was approximately 95%, and mainly papillary thyroid tumours were induced. For elucidating the molecular event in Rivulus papillary thyroid carcinogenesis, we first analysed for ras gene mutations based on the known ability of MNU to induce point mutations. The amplified R. Marmoratus Ha-and Ki-ras gene exon 1 and 2 regions were checked along with confirming the presence and expression patterns of the DNA repair gene O(6)-methylguanine alkyltransferase (O(6)-MT) and other oncogenes (c-src, c-fos, and c-myc). Ha- and Ki-ras genes from 38 tumour samples were tested for point mutations with direct sequencing but were not found to contain mutations. In this report, based on the lack of Ha- and Ki-ras gene mutations in papillary thyroid tumour induction in R. Marmoratus, we suggest that Ha- and Ki-ras gene-independent pathways such as ret/PTC rearrangements and other activations are involved in Rivulus papillary thyroid carcinogenesis. Teratogenesis Carcinog. Mutagen. 20:1-9, 2000.

Absence of the c-Ha-ras and c-Ki-ras oncogene mutations in the hermaphroditic fish Rivulus marmoratus papillary thyroid carcinomas induced by N-methyl-N'-nitro-N-nitrosoguanidine

Previously we reported that papillary thyroid carcinomas were predominantly induced at high frequency by a low dose of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in the hermaphroditic fish Rivulus marmoratus. In the current study, polymerase chain reaction (PCR) amplification and direct sequencing were used to examine the point mutations of Ha- and Ki-ras genes, which may be associated with papillary thyroid tumour development in rivulus. Thirty-three tumour samples were tested, however, no mutations were detected in rivulus Ha- and Ki-ras genes. In human and rodent models, it has been reported that ras gene mutations in papillary thyroid tumours occurred preferentially in the N-ras gene in general, while follicular tumours contained activated Ha- or Ki-ras gene mutations. This may explain why papillary thyroid carcinomas in rivulus were not mutated at codon 12, 13 or 61 of exon 1 or exon 2 of the rivulus Ha- or Ki-ras gene. These results imply that another oncogene, such as the N-ras gene and others, may be preferentially activated in rivulus papillary thyroid carcinomas, and also give valuable information for comparative studies of papillary thyroid carcinogenesis.

Phenotypic plasticity in the growth of the self-fertilizing hermaphroditic fish Rivulus marmoratus

Temperature, salinity, food ration and interactions among them had significant effects on overall growth of hatchlings of the self-fertilizing hermaphrodite Rivulus marmoratus. Low temperature (19° C) suppressed growth. At 26° C, the treatment groups at 12 and 40‰ salinity had higher final wet masses than the group at 1‰. When provided with high food ration, individuals whose parents were provided with low food had a significantly higher specific growth rate in the first month than those whose parents were provided with high food ration. This effect was also found in the final wet masses. It appears that genetic variation in this species occurs only between strains, yet within a strain there is considerable scope for phenotypic plasticity. The combination of a fixed genotype and plastic expression, along with a suite of specialized physiological characteristics, may explain how a self-fertilizing hermaphrodite can thrive in such an abiotically stressful and variable habitat as an estuary.

Phenotypic plasticity in the growth of the self-fertilizing hermaphroditic fishRivulus marmoratus

Phenotypic plasticity in the growth of the self-fertilizing hermaphroditic fishRivulus marmoratus

Age Composition in the Schools of Juvenile Yellowtail <i>Seriola quinqueradiata</i> Associated with Drifting Seaweeds in the East China Sea

Title Age, Growth, and Sexual Development in the Self-fertilizing Hermaphroditic Fish Rivulus marmoratus Author(s) Sakakura, Yoshitaka; Noakes, David L. G. Citation Environmental Biology of Fishes, 59(3), pp.309-317; 2000 Issue Date 2000-11 URL http://hdl.handle.net/10069/35743 Right © 2000 Kluwer Academic Publishers. Printed in the Netherlands.; The final publication is available at link.springer.com NAOSITE: Nagasaki University's Academic Output SITE

Age-related thyroid tumor induction by N-methyl-N'-nitro-N-nitrosoguanldlne on the hermaphroditic fish rivulus marmoratus

Age-related thyroid tumor induction by N-methyl-N'-nitro-N-nitrosoguanldlne on the hermaphroditic fish rivulus marmoratus

Scale Growth and Squamation Chronology for the Laboratory-Reared Hermaphroditic Fish Rivulus marmoratus (Cyprinodontidae)

Scale morphology, growth and the squarnation chronology are described for the hermaphroditic fish Rivulus marmoratus reared in the laboratory. The scales are round or oval shaped cycloid type, and their sizes are about 0.3–1.0 mm in diameter. The number of ridges increases more rapidly relative to the body growth of the fish in early stages, but this increase is proportionate to growth subsequently. Three loci of scale development have been identified. The scales first appeared on the center of the parietal region at 8 days after hatching. The second locus of scale formation was on the lateral line of the posterior end of the caudal peduncle. A third locus was later observed on the lower right corner of the operculum: The final squamation was completed at 6 weeks after hatching.

Hepatocarcinogenicity of Diethylnitrosamine to the Self-Fertilizing Hermaphroditic Fish <italic>Rivulus marmoratus</italic> (Teleostomi: Cyprinodontidae)<xref ref-type="fn" rid="FN1">2</xref>

The larvae, 3 days post hatching, of self-fertilizing hermaphroditic fish Rivulus marmoratus were exposed to diethylnitrosamine (DENA) (CAS: 55-18-5; N-nitrosodiethylamine) at levels of 50-400 ppm for 2 hours. At 3-month intervals, the exposed fish were sampled until 9 months postcarcinogen treatment. Hepatomas were induced in 6- and 9- month groups of the 100-ppm-treated and 200-ppm-treated fish. An exposure of fish larvae to 400 ppm DENA also produced similar tumors on 3-, 6-, and 9-month groups. The tumor incidences were clearly dose dependent. These results offered the possibility of the use of R. marmoratus as a suitable model for experimental carcinogenesis.

Delimitation of the Thermolabile Phenocritical Period of Sex Determination and Differentiation in the Ontogeny of the Normally Hermaphroditic Fish Rivulus marmoratus Poey

Delimitation of the Thermolabile Phenocritical Period of Sex Determination and Differentiation in the Ontogeny of the Normally Hermaphroditic Fish Rivulus marmoratus Poey

The Homozygosity of Clones of the Self-Fertilizing Hermaphroditic Fish Rivulus marmoratus Poey (Cyprinodontidae, Atheriniformes)

Two clones of the hermaphroditic fish Rivulus marmoratus Poey, identified by their intraclonal histocompatibilities and interclonal histoincompatibility, were kept through successive uniparental generations, each fish in isolation. These hermaphrodites emit internally self-fertilized eggs incubated up to 2 1/2 days intraparentally and thereafter extraparentally. By extraparental incubation at low temperature, primary males can be produced at will from eggs otherwise yielding hermaphrodites. Secondary males originate from hermaphrodites, usually late in life, if the ovarian component of the ovotestis involutes. There are no females. Eggs from an aberrant hermaphrodite chronically emitting un-self-fertilized eggs were cross-fertilized artificially and also naturally by sperm from a primary male. A fin graft from the clone of the male parent was accepted (autograft reaction) by the interclonal hybrid, showing that amphimixis had occurred and that the clone of the graft donor is homozygous for the histocompat...

STAGES IN THE NORMAL DEVELOPMENT OF FUNDULÚS HETEROCLITUS

Next article No AccessJournal ArticlesSTAGES IN THE NORMAL DEVELOPMENT OF FUNDULÚS HETEROCLITUSPHILIP B. ARMSTRONG and JULIA SWOPE CHILDPHILIP B. ARMSTRONGMarine Biological Laboratory, Woods Hole, Mass. 02543State University of New York, College of Medicine, Syracuse, New York 13210 Search for more articles by this author and JULIA SWOPE CHILDMarine Biological Laboratory, Woods Hole, Mass. 02543State University of New York, College of Medicine, Syracuse, New York 13210 Search for more articles by this author Marine Biological Laboratory, Woods Hole, Mass. 02543State University of New York, College of Medicine, Syracuse, New York 13210PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 128, Number 2April 1965 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.2307/1539545 Views: 245Total views on this site Citations: 2Citations are reported from Crossref © 1965 Marine Biological LaboratoryPDF download Crossref reports the following articles citing this article: Leonard DiMichele , and Dennis A. Powers Developmental and Oxygen Consumption Rate Differences between Lactate Dehydrogenase-B Genotypes of Fundulus heteroclitus and Their Effect on Hatching Time, Physiological Zoology 57, no.11 (Sep 2015): 52–56.https://doi.org/10.1086/physzool.57.1.30155967 Robert W. Harrington Jr. Delimitation of the Thermolabile Phenocritical Period of Sex Determination and Differentiation in the Ontogeny of the Normally Hermaphroditic Fish Rivulus marmoratus Poey, Physiological Zoology 41, no.44 (Sep 2015): 447–460.https://doi.org/10.1086/physzool.41.4.30155480