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.e. five exons interrupted by four introns) compared to most other teleostean Aqp1ab orthologues, which have four exons. The 5'-flanking regulatory region of Aqp gene showed diverse transcription factor binding motifs, particularly those associated with stress/immune responses. Developmental expression patterns indicated that Aqp1ab mRNA was maternally inherited, presumably important for fine-tuning gene expression during embryonic and early larval developments. Expression of mud loach Aqp1ab mRNA was significantly and differentially modulated in several tissues (intestine, kidneys, spleen, and liver) in response to various heavy metal treatments. In addition, Aqp1ab gene expression was highly induced in response to immune challenge (LPS and polyI:C injections). Collectively, our results suggested that AQPs are multifunctional effectors playing diverse roles in cellular pathways relevant to immune and/or stress adaptation responses, in addition to their involvement in osmoregulation.

Transcriptome Analysis of Maternal Gene Transcripts in Unfertilized Eggs of Misgurnus anguillicaudatus 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, Misgurnus anguillicaudatus, 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. Comparison expression profiles and analysis of tissue specificity revealed that the large numbers of maternal transcripts were stored in unfertilized eggs near the late phase of ovarian maturation and before ovulation. Functional classification showed a total of 279 maternal immune-related transcripts classified with immune system process GO term and immune system KEGG pathway. qPCR analysis showed that transcript levels of identified maternal immune-related candidate genes were dynamically modulated during development and early ontogeny of M. anguillicaudatus. Taken together, this study could not only provide knowledge on the protective roles of maternal immune-related genes during early life stage of M. anguillicaudatus but could also be a valuable transcriptomic/genomic resource for further analysis of maternally provisioned genes in M. anguillicaudatus and other related teleost fishes.

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.e., tripartite organization) and peptide (two conserved disulfide bonds) levels. A. baerii LEAP2 isoforms (designed LEAP2AB and LEAP2C, respectively) phylogenetically occupy the most basal position in the actinopterygian lineage and represent an intermediate character between teleostean and tetrapodian LEAP2s in the sequence alignment. Molecular phylogenetic analysis including LEAP2s from extant primitive fish species indicated that the evolutionary origin of ancestral LEAP2 in vertebrate groups should date back to earlier than the actinopterygian-sarcopterygian split. Gene expression assays under both basal and stimulated conditions suggested that A. baerii LEAP2 isoforms have undergone substantial subfunctionalization in tissue distribution pattern, developmental/ontogenetic expression, and immune responses. LEAP2AB showed a predominant liver expression, while LEAP2C exhibited the highest level of expression in the intestine. LEAP2C was a more dominantly expressed isoform during embryonic development and prelarval ontogeny. The LEAP2AB isoform is more closely associated with innate immune response to microbial invasion, compared with LEAP2C, as evidenced by results from LPS, poly(I:C) and Aeromonas hydrophila challenges. Synthetic mature peptides of LEAP2AB displayed a more potent antimicrobial activity than did LEAP2C. Data from this study could be useful not only to provide deeper insights into the evolutionary mechanism of LEAP2 in the actinopterygian lineage but also to better understand the innate immunity of this commercially important chondrostean species.

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. Here, we first report potential a prototype hepcidin from the Siberian sturgeon Acipenser baerii, a primitive chondrostean species. The A. baerii hepcidin (AbHAMP) gene showed a tripartite exon-intron organization, which encoded a precursor protein comprised of three structural signatures containing eight cysteine residues, a common structure in vertebrate hepcidin genes and proteins. mRNA expression by iron-overloading and bacterial infection and antibacterial activity revealed that AbHAMP might play a role in iron metabolism regulator in the liver, and in direct and/or indirect host immune response in the kidney against invading pathogen. Comparison of gene and protein sequences reveled that AbHAMP possesses intermediate characteristics between tetrapodian and teleostean hepcidins (HAMP1s). Phylogenetically, AbHAMP had a closer genetic affiliation to tetrapodian orthologs than to teleostean orthologs, suggesting that the structures of this chondrostean hepcidin may closely reflect the structures of an evolutionarily ancestral form that might have evolved into extant hepcidins in tetrapods and teleosts, respectively. Based on the identification of hepcidin from the chondrostean group, the emergence of the common ancestral hepcidin should be traced back to in early Osteichthyes: no later than sarcopterygian (lobe-finned fishes) - actinopterygian (ray-finned fishes) split.

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. Among them, a mussel-inspired polymer coating has been extensively utilized owing to its wide applicability. For instance, adhesion of human embryonic stem cells and neuronal cells on solid surfaces has been improved. Despite the excellent capability of the mussel-inspired polymer coating, most studies have primarily focused on mammalian cells. However, the efficacy of these coatings on the adhesion of other cell lines is yet unclear. This study aimed to assess the potential of the mussel-inspired polymer coating in the regulation of the adhesion of fish ovarian germline stem cells on solid surfaces. Solid surfaces were coated by polydopamine and poly-L-lysine, and the effect of the coatings on cellular behaviors was investigated.

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, Isochrysis galbana, Pavlova viridis, and Chlamydomonas coccoides. Both of the IBPs showed IR inhibition in f/2 medium containing 10% DMSO, indicating that they retain their function in freezing media. Microalgal cells were frozen in 10% DMSO with or without IBP. Post-thaw viability exhibited that the supplementation of IBPs increased the viability of all cryopreserved cells. LeIBP was effective in P. viridis and C. coccoides, while FfIBP was in I. galbana. The cryopreservative effect was more drastic with P. viridis when 0.05 mg/mL LeIBP was used. These results clearly demonstrate that IBPs could improve the viability of cryopreserved microalgal cells.

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.e., tripartite organization) and amino acid (conserved Cys motifs) levels. The 5'-flanking regulatory region of abalone MT gene displayed various transcription factor binding motifs particularly including ones related with metal regulation and stress/immune responses. Tissue distribution and basal expression patterns of MT mRNAs indicated a potential association between ovarian MT expression and sexual maturation. Developmental expression pattern suggested the maternal contribution of MT mRNAs to embryonic and early larval developments. Abalone MT mRNAs could be significantly induced by various heavy metals in different tissues (gill, hepatopancreas, muscle and hemocyte) in a tissue- and/or metal-dependent fashion. In addition, the abalone MT gene was highly modulated in responsive to other non-metal, stimulatory treatments such as immune challenge (LPS, polyI:C and bacterial injections), hypoxia (decrease from normoxia 8 ppm-2 ppm), thermal elevation (increase from 20 °C to 30 °C), and xenobiotic exposure (250 ppb of 17α-ethynylestradiol and 0.25 ppb of 2,3,7,8-tetrachlorodibenzodioxin) where differential expression patterns were toward either up- or down-regulation depending on types of stimulations and tissues examined. Taken together, our results highlight that MT is a multifunctional effector playing in wide criteria of cellular pathways especially associated with development and stress responses in this abalone species.

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. We observed both clonogenicity and AP activity in all five sub-cell lines, but the proportion of cells that exhibited both properties was significantly different among them. Even though we detected different formation rates and sizes of embryoid body (EB) among these cells, all lines were stably able to form EBs and further induction for differentiation showed their capability to differentiate into other cell types in a spontaneous manner. From this study, we determined that the embryonic cell lines examined possessed heterogeneous ESC activities and can be utilized as a marine model system for fish ESC-based research.

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. Both rbWap65 genes were predicted to possess various transcription factor (TF) binding motifs related with stress and innate immunity in their 5ʹ-upstream regions, in which inflammation-related motifs were more highlighted in the rbWap65-2 than in rbWap65-1. Based on the RT-PCR assay, the liver-predominant expression pattern was more apparent in rbWap65-1 than rbWap65-2 isoform. During thermal elevation, clear upregulation was found only for the rbWap65-1. In contrast, immune stimulations (bacterial challenges, viral infection and iron overload) activated more preferentially the rbWap65-2 isoform in overall, although the inducibility was affected by the kinds of stimulators and tissue types. Taken together, our data suggest that the two paralogue rbWap65 isoforms have experienced subfunctionalization and/or neofunctionalization during their evolutionary history, in which the rbWap65-2 has retained closer, functional orthology to the human HPX while the rbWap65-1 have been diversified to be more related with thermal acclimation physiology.

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. Although asparagine-proline-alanine (NPA) motifs are highly conserved in most odAQP isoforms, several AQPs revealed variant types of motifs such as asparagine-proline-proline (NPP), asparagine-proline-valine (NPV) or/and asparagine-proline-serine (NPS) motifs. The phylogenic analysis showed that marine medaka AQPs had closet relationship with Japanese ricefish (medaka; Oryzias latipes) counterparts. Reverse transcription (RT)-PCR analyses showed that marine medaka AQP transcripts would be expressed in not only osmoregulatory tissues but also nonosmoregulatory tissues, and also that the expression levels of certain AQP isoforms in nonosmoregulatory tissues were readily comparable or even higher than those in typically known osmoregulatory organs. Although the overall tissue distribution patterns of AQPs were not significantly different between 0- and 30-ppt acclimated fish, the expression levels under different salinities were largely variable among isoforms and tissues. This is the first report to investigate tissue expression profiles of teleostean AQPs 11 and 12 during the long-term acclimation to freshwater and salted water.

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-17ß (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.

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. In adult transgenics, an authentic brilliant red fluorescence was achieved over the skeletal muscles of the transgenic individuals, which might be sufficient for ornamental display. Expression analysis of the transgenic mRNAs indicated that rfp transcripts were predominantly expressed in the skeletal muscles. Different transgenic lines displayed different levels of transgene expression at the mRNA, protein, and phenotypic levels. However, the efficiency of transgene expression was independent of the transgene copy number. The RFP protein levels were consistently stable in the transgenic fish muscles through several generations, up to F5. The results of this study suggest that transgenic marine medaka that acquire strong fluorescent signals in their skeletal muscles can be developed as a promising, novel ornamental fish for display in both freshwater and seawater aquaria.

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. In overall, mlWap65-2 is more homologous to human hemopexin than is mlWap65-1. Both mud loach Wap65 transcripts are predominantly expressed in liver, although the transcripts are ubiquitously detectable in most tissues with variable basal expression. Both mlWap65 isoforms are differentially regulated during embryonic development, and the changes in transcript levels during embryogenesis are greater for mlWap65-2 than for mlWap65-1. The transcription of the mlWap65 genes is differentially modulated by various stimuli, including thermal changes, immune challenge (lipopolysaccharide injection or bacterial infection), and heavy metal exposure (cadmium, copper, or nickel). The isoform mlWap65-1 is more responsive to warm temperature treatments than mlWap65-2, whereas mlWap65-2 is much more strongly stimulated by immune and heavy metal challenges than is mlWap65-1. Taken together, the results of this study suggest that mud loach Wap65 isoforms are potentially involved in multiple cellular pathways and that the two mud loach Wap65 isoforms undergo functional partitioning or subfunctionalization.

Functional ability of cytoskeletal ß-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena.

Marine medaka Oryzias dancena, a candidate model organism, represents many attractive merits as a material for experimental transgenesis and/or heterologous expression assay particularly in the field of ecotoxicology and developmental biology. In this study, cytoskeletal ß-actin gene was characterized from O. dancena and the functional capability of its promoter to drive constitutive expression of foreign reporter protein was evaluated. The O. dancena ß-actin gene possessed a conserved genomic organization of vertebrate major cytoplasmic actin genes and the bioinformatic analysis of its 5'-upstream regulatory region predicted various transcription factor binding motifs. Heterologous expression assay using a red fluorescent protein (RFP) reporter construct driven by the O. dancena ß-actin regulator resulted in stunningly bright expression of red fluorescence signals in not only microinjected embryos but also grown-up transgenic adults. Although founder transgenics exhibited mosaic patterns of RFP expression, transgenic offspring in subsequent generations displayed a vivid and uniform expression of RFP continually from embryos to adults. Based on the blot hybridization assays, two transgenic lines established in this study were proven to possess high copy numbers of transgene integrants (approximately 240 and 34 copies, respectively), and the transgenic genotype in both lines could successfully be passed stably up to three generations, although the rate of transgene transmission in one of the two transgenic lines was significantly lower than expected Mendelian ratio. Significant red fluorescence color could be ubiquitously observable in all the tissues or organs of the transgenics. Quantitative real-time RT-PCR represented that the expression pattern of transgene under the regulation of ß-actin promoter would resemble, in overall, the regulation of endogenous ß-actin gene in adult tissues, although putative mechanism for competitive or independent regulation between transgene and endogenous gene could also be found in several tissues. Results from this study undoubtedly indicate that the O. dancena ß-actin promoter would be powerful enough to fluorescently visualize most cell types in vivo throughout its whole lifespan. This study could be a useful start point for a variety of transgenic experiments with this species concerning the constitutive expression of living fluorescent color reporters and other foreign proteins.

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. The two alleles are transmitted from parental fish to offspring with a Mendelian inheritance pattern, as demonstrated with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping. Southern blot hybridization analysis showed a high degree of polymorphisms in the restriction patterns of individuals. Mud loach hamp1 mRNA is predominantly expressed in the liver, although many other tissues showed detectable levels of hamp1 transcripts in RT-PCR assay. Lipopolysaccharide and bacterial challenges induced significant hamp1 expression, whereas hamp1 was not clearly stimulated by polyinosinic:polycytidylic acid [poly(I:C)] injection. Iron overload and Cu exposure also elevated hamp1 transcripts in various tissues. The transcriptional activation of mud loach hamp1 in response to these stimuli varied among tissue types, and the liver appears predominantly involved in hepcidin-mediated iron regulation. However, hepcidin expression in the kidney and spleen was preferentially modulated by inflammation-mediated signals produced by immune challenges. Our results suggest that mud loach hepcidin has two basic functions, in iron regulation and antimicrobial activity, and that its transcription is also modulated by other environmental perturbations, including heavy metal exposure.

Superoxide Dismutase Multigene Family from a Primitive Chondrostean Sturgeon, Acipenser baerii: 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, Acipenser baerii, enabling the comparison of their transcriptional regulation patterns during development, prelarval ontogeny, and immune stimulation. Each A. baerii 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. The expression of the AbSOD isoforms showed isoform-dependent dynamic modulation according to embryonic development and prelarval ontogenic behaviors. Prelarval microinjections revealed that lipopolysaccharide only induced AbSOD3 expression, while Aeromonas hydrophila induced the expression of AbSOD2 and AbSOD3. In fingerlings, the transcriptional response of each AbSOD isoform to bacterial infection was highly tissue-specific, and the three isoforms exhibited different response patterns within a given tissue type; AbSOD3 was induced the most sensitively, and its induction was the most pronounced in the kidneys and skin. Collectively, these findings suggest isoform-dependent roles for the multigene SOD family in antioxidant defenses against the oxidative stress associated with development and immune responses in these endangered sturgeon fish.

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. A total of 316 million clean reads were de novo assembled into 271,039 contigs, of which a transcriptome with 209,974 non-redundant transcripts was produced. Using generalized fold change (GFOLD) algorithm with a cut-off │GFOLD value│ > 4, we identified differentially expressed transcripts (DETs) from diploid and triploid abalone in responses to acute heat-stress and hypoxia treatments, respectively. Comparative analysis of the identified DETs revealed alteration of transcript expression profile, level, and process in triploid abalone compared to their diploid siblings. Thus, our study will provide not only comprehensive insight into understanding of the transcriptional regulation to environmental stresses in triploid abalone but a framework for efficient management of triploid abalone aquaculture.

Development of siRNA expression vector utilizing rock bream beta-actin promoter: a potential therapeutic tool against viral infection in fish.

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

Variability of responses in the crucian carp Carassius carassius from two Ukrainian ponds determined by multi-marker approach.

The aim of this study was to evaluate the health status of the comparatively tolerant fish Carassius carassius over three seasons in an area characterized by spontaneous human activities. Sites near the springs of a river (site Z) and downstream of a river (site B) in Western Ukraine were selected. According to the centroid grouping analysis, the biochemical and morphological indices allowed the fish to be distinguished according to season more than to site. The level of nuclear abnormalities was low in fish from both sites. However C. carassius inhabiting site B showed a lower metal-binding capacity of MTs in relation to fish from site Z. This was combined with high levels of MT protein (particularly in the liver), and reduced glutathione (GSH) and redox state of GSH (particularly in the gills), which might confer some advantages to fish inhabiting this site. The levels of ethoxyresorufin-O-deethylase, glutathione-S-transferase, cholinesterase and vitellogenin-like proteins indicated significant but intermittent inter-site differences. In summer, oxidative damage due to a high level of lipid peroxidation, and low superoxide dismutase and catalase activities was observed in fish from site B, and in autumn, it was observed in the gills of fish from site Z. The relationship between MT protein levels and antioxidant defense and the lack of a positive relationship between MT levels and their metal-binding capacity was confirmed by principal component analysis.

Generation of Vibrio anguillarum ghost by coexpression of PhiX 174 lysis E gene and staphylococcal nuclease A gene.

Vibrio anguillarum ghosts (VAG) were generated, for the first time, using a conjugation vector containing a ghost bacteria inducing cassette, pRK-lambdaP(R)-cI-Elysis, in which the expression of PhiX174 lysis gene E was controlled by the P ( R )/cI regulatory system of lambda phage. By scanning electron microscopy, holes ranging 80-200 nm in diameter were observed in the VAG. To avoid the presence of bacterial genomic DNA and an antibiotic resistance gene in the final VAG product, we constructed a new dual vector, pRK-lambdaP(R)-cI-E-SNA, containing the E-mediated lysis cassette and the staphylococcal nuclease A (SNA)-mediated DNA degradation cassette, and generated safety-enhanced VAG for use as a fish vaccine.