Characterization of aquaporin-1ab (Aqp1ab) mRNA in mud loach (Misgurnus mizolepis) exposed to heavy metal and immunostimulant stimuli.

Aquaporins (AQPs) are key proteins that regulate fluid homeostasis in cells via modulating osmotic water transport. In the present study, we identified three variants of Aqp1ab transcript (mmAQP1ab x1, mmAQP1ab x2, and mmAQP1ab x3) in mud loaches (Misgurnus mizolepis), and their expression patterns were examined in response to heavy metal and immunostimulant exposure. Mud loach Aqp1ab gene has a somewhat different organizational structure (i.

Transcriptome expression profiles between diploid and triploid Pacific abalone (Haliotis discus hannai) juveniles in response to acute heat-stress and hypoxia treatments.

With an increasing interest for the use of triploids in abalone aquaculture, it is crucial to understand their physiological responses to environmental stress, particularly such as heat-stress and hypoxia, which are significant factors that cause adverse effects on the efficiency and capacity of farming practice in abalone production. However, nothing is known about gene expression of triploid abalone to modulate physiological responses under different environmental stresses. Transcriptomic response to the acute heat-stress and hypoxia were explored in hepatopancreas of diploid and triploid Pacific abalone (Haliotis discus hannai) juveniles.

Superoxide Dismutase Multigene Family from a Primitive Chondrostean Sturgeon, : Molecular Characterization, Evolution, and Antioxidant Defense during Development and Pathogen Infection.

Three distinct superoxide dismutases (SODs)-copper/zinc-SOD (SOD1), manganese-SOD (SOD2), and extracellular copper/zinc-SOD (SOD3)-were identified from a primitive chondrostean fish, , enabling the comparison of their transcriptional regulation patterns during development, prelarval ontogeny, and immune stimulation. Each SOD isoform (AbSOD) shared conserved structural features with its vertebrate orthologs; however, phylogenetic analyses hypothesized a different evolutionary history for AbSOD3 relative to AbSOD1 and AbSOD2 in the vertebrate lineage. The AbSOD isoforms showed different tissue distribution patterns; AbSOD1 was predominantly expressed in most tissues.

Transcriptome Analysis of Maternal Gene Transcripts in Unfertilized Eggs of and Identification of Immune-Related Maternal Genes.

Maternal genes are important in directing early development and determining egg quality in fish. We here report the de novo transcriptome from four tissue libraries of the cyprinid loach, , and for the first time identified maternal gene transcripts in unfertilized eggs and suggest their immune system involvement. Expression profiles and functional enrichment revealed a total 24,116 transcripts were expressed as maternal transcripts in unfertilized eggs, which were involved in a wide range of biological functions and pathways.

Transcriptional Activity of an Estrogen Receptor β Subtype in the Medaka .

In vertebrate reproductive system, estrogen receptor (ER) plays a pivotal role in mediation of estrogenic signaling pathways. In the present study, we report the cDNA cloning, expression analysis, and transcriptional activity of ERβ1 subtype from medaka . The deduced ERβ1 (odERβ1; 519 amino acids) contained six characteristic A/B to E/F domains with very short activation function 2 region (called AF2).

Characterization of testis-specific serine/threonine kinase 1-like (TSSK1-like) gene and expression patterns in diploid and triploid Pacific abalone (Haliotis discus hannai; Gastropoda; Mollusca) males.

Testis-specific serine/threonine kinase 1-like (TSSK1-like), which plays important roles in late-phase spermatogenesis and male fertility, was characterized in Pacific abalone Haliotis discus hannai, an important commercial marine gastropod. Further, its expression patterns were assessed in diploid and induced triploid males showing differential degrees of testis maturation. Abalone TSSK1-like shared conserved structural features with mammalian TSSK1s and other potential metazoan orthologs, especially regarding the catalytic STKc domain.

Subfunctionalization and evolution of liver-expressed antimicrobial peptide 2 (LEAP2) isoform genes in Siberian sturgeon (Acipenser baerii), a primitive chondrostean fish species.

Two liver-expressed antimicrobial peptide 2 (LEAP2) isoforms were characterized in a primitive chondrostean sturgeon species, Acipenser baerii (Acipenseriformes). A. baerii LEAP2 isoforms represented essentially common structures shared by their vertebrate orthologs at both genomic (i.

Chondrostean sturgeon hepcidin: An evolutionary link between teleost and tetrapod hepcidins.

Hepcidin, a cysteine-rich antimicrobial peptide (AMP), plays key roles as a regulatory hormone in iron homeostasis, providing a link between iron metabolism and innate immunity. Unlike many other AMPs displaying a high degree of sequence variability among closely related organisms, hepcidin is highly conserved from teleosts to mammals. However, little is known about the early ancestry of hepcidins in the vertebrate lineage.

Gene delivery into Siberian sturgeon cell lines by commercial transfection reagents.

The optimal transfection conditions for efficient transgene delivery into a specific cell type should be empirically determined, particularly in cases involving unusual cell types. We compared the conditions for effective introduction of transgenes into Siberian sturgeon (Acipenser baerii) cell lines by evaluating the cytotoxicity and transfection efficiency of three commercially available transfection reagents: Lipofectamine 2000, X-tremeGENE HP DNA Transfection Reagent, and GeneJuice Transfection Reagent. Plasmid vectors containing the gene encoding enhanced green fluorescent protein were mixed with each of the transfection reagents using reagent-to-plasmid ratios of 1:1, 2:1, and 4:1.

Anesthetic protocol for microinjection-related handling of Siberian sturgeon (Acipenser baerii; Acipenseriformes) prolarvae.

An anesthetic protocol was optimized for microinjection-related handling of Siberian sturgeon (Acipenser baerii; Acipenseriformes) prolarvae, an extant primitive fish species commonly grown in aquaculture. Comparative examinations of three selected anesthetics (clove oil, lidocaine, and MS-222) with a dosage regime of 50, 100, 200, and 400 mg/L indicated that MS-222 was the most efficient agent for Siberian sturgeon prolarvae, as evidenced by the fast induction of anesthesia with quick and uniform recovery. Meanwhile, clove oil should be avoided, due to prolonged recovery times varying widely between individuals.

Enhanced Adhesion of Fish Ovarian Germline Stem Cells on Solid Surfaces by Mussel-Inspired Polymer Coating.

Development of advanced cell culture methods has gained increasing attention because it allows for efficient genetic engineering and precise regulation of animal reproduction on a cellular basis. Numerous studies have attempted to develop an advanced cell culture method. Previous studies have altered cell culture media and pretreated culture plates with functional molecules.

Non-lethal method for the preparation of metaphase spreads using cultured mantle tissue from live adult abalone.

Metaphase spread preparation in adult abalone has not been successful, which has restricted the applications of karyotyping-based technologies. Here, we present a non-lethal method to enable preparation of metaphase spreads from live adult abalone using a tissue culture method. Mantle tissue fragments from live adult abalone were cultured in vitro and the cultured cells were used for metaphase spread preparation.

Effect of Marine-Derived Ice-Binding Proteins on the Cryopreservation of Marine Microalgae.

Ice-binding protein (IBPs) protect cells from cryo-injury during cryopreservation by inhibiting ice recrystallization (IR), which is a main cause of cell death. In the present study, we employed two IBPs, one, designated LeIBP from Arctic yeast, and the other, designated FfIBP from Antarctic sea ice bacterium, in the cryopreservation of three economically valuable marine microalgae, , and . Both of the IBPs showed IR inhibition in f/2 medium containing 10% DMSO, indicating that they retain their function in freezing media.

Effects of Clove Oil and Lidocaine-HCl Anesthesia on Water Parameter during Simulated Transportation in the Marine Medaka, .

Optimum concentrations of anesthetic clove oil and anesthetic lidocaine-HCl were determined for a species of adult marine medaka, , over a range of salinity conditions, and investigated in a transport simulation experiment by analyzing various water and physiological parameters. Research indicated that the higher the concentration of anesthetic at each salinity, the shorter the anesthesia time at each salinity. At each concentration, fish were anesthetized slower at water salinities over 10 ppt (<0.

Comparative Study of Growth and Gonad Maturation in Diploid and Triploid Marine Medaka, .

The marine medaka, is a suitable sample as a laboratory animal because it has a small size and clearly distinguishes between female and male. Data on the growth and maturity of the diploid and triploid sea cucurbit species suitable for laboratory animals are very useful for studying other species. Triploidy was induced in the marine medaka by cold shock treatment (0°C) of fertilized eggs for 45 min, applied two minutes after fertilization.

Transcriptional responses of metallothionein gene to different stress factors in Pacific abalone (Haliotis discus hannai).

A novel metallothionein (MT) gene from the Pacific abalone H. discus hannai was characterized and its mRNA expression patterns (tissue distribution, developmental expression and differential expression in responsive to various in vivo stimulatory treatments) were examined. Abalone MT shares conserved structural features with previously known gastropod orthologs at both genomic (i.

Identification of embryonic stem cell activities in an embryonic cell line derived from marine medaka (Oryzias dancena).

This study was conducted to identify embryonic stem cell (ESC) activities of a long-term cultured embryonic cell line previously derived from blastula-stage Oryzias dancena embryos. Five sub-cell lines were established from the embryonic cell line via clonal expansion of single cells. ESC activities, including clonogenicity, alkaline phosphatase (AP) activity, and differentiation capacity, were examined in the five sub-cell lines.

Establishment condition and characterization of heart-derived cell culture in Siberian sturgeon (Acipenser baerii).

This study was conducted to establish the efficient condition for stable derivation of heart-derived cell culture in Siberian sturgeon (Acipenser baerii). Three factors including isolation methods, cell densities in initial seeding, and basal media were evaluated for the derivation of heart-derived cell culture. As the results, enzymatic isolation was more efficient than mechanical isolation in both cell retrieval and further culture.

Isolation and mRNA expression analysis of aquaporin isoforms in marine medaka Oryzias dancena, a euryhaline teleost.

We have identified six putative aquaporin (AQP) genes from marine medaka Oryzias dancena (named odAQPs 1, 3, 8, 10, 11 and 12). The marine medaka AQP cDNAs encode polypeptides of 259-298 amino acids, respectively. Topology predictions showed six transmembrane domains, five connecting loops, and cytoplasmic N- and C-terminal domains, all of which is conserved among AQP molecules.

Genomic organization and functional diversification of two warm-temperature-acclimation-associated 65-kDa protein genes in rockbream (Oplegnathus fasciatus; Perciformes).

Two paralogue genes of warm-temperature-acclimation-associated 65-kDa protein were characterized and their mRNA expression patterns during various experimental stimulations were examined in the rockbream (Oplegnathus fasciatus; Perciformes). Rockbream Wap65 isoforms (rbWap65-1 and rbWap65-2) share basically common structural features with other teleostean orthologues and human hemopexin (HPX) at both amino acid (conserved cysteine and histidine residues) and genomic levels (ten-exon structure), although the rbWap65-2 reveals more homologous characteristics to human HPX than does rbWap65-1 isoform. Southern blot analysis indicates that each rbWap65 isoform exists as a single copy gene in the rockbream genome.

Characterization of stable fluorescent transgenic marine medaka (Oryzias dancena) lines carrying red fluorescent protein gene driven by myosin light chain 2 promoter.

Stable transgenic germlines carrying the red fluorescence protein (RFP) gene (rfp) driven by fast skeletal myosin light chain-2 gene (mlc2f) promoter were established in a truly euryhaline fish species, the marine medaka (Oryzias dancena; Beloniformes). Transgenic lines contained transgene copy numbers varying from a single copy to more than 230 copies per genome. Although the transgenic founders displayed mosaic and/or ectopic expression of the RFP signal, the resultant F1 transgenics and their progeny showed consistently stable transmission of the transgenic locus and uniform RFP signal through several subsequent generations.

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.

Modulation of warm-temperature-acclimation-associated 65-kDa protein genes (Wap65-1 and Wap65-2) in mud loach (Misgurnus mizolepis, Cypriniformes) liver in response to different stimulatory treatments.

Two paralogous isoform cDNAs of warm-temperature-acclimation-associated 65-kDa protein (Wap65-1 and Wap65-2) were isolated from the cypriniform species, mud loach (Misgurnus mizolepis), and characterized. The deduced amino acid sequences of the two mud loach Wap65 isoforms (mlWap65-1 and mlWap65-2) share moderate levels of sequence homology with their corresponding orthologues from teleosts and with human hemopexin, a possible mammalian homologue. Both isoforms display conserved features, including essential motifs and/or residues that are important for the protein structure of hemopexin.

Molecular characterization of hepcidin gene from mud loach (Misgurnus mizolepis; Cypriniformes).

The gene encoding hepcidin, an antimicrobial peptide, was isolated and characterized in the mud loach Misgurnus mizolepis (Cypriniformes). Mud loach hepcidin shows a considerable degree of structural homology to other vertebrate hamp1 orthologues at both the gene and protein levels, particularly with respect to its tripartite genomic organization, typical transcription-factor-binding motifs in its promoter, and conserved cysteine residues in the mature cationic peptide. The mud loach possesses at least two allelic forms of hamp1, which are expected to be translated into the same hepcidin preproprotein.

Various responses to copper and manganese exposure of Carassius auratus gibelio from two populations.

The aim of this study was to compare the effect of pro-oxidants copper (Cu(2+), 0.005 and 0.050mg L(-1)) or manganese (Mn(2+), 0.