Molecular cloning, expression analysis of interleukin 17D (cysteine knot cytokine) from Amphiprion clarkii and their functional characterization and NFκB pathway activation using FHM cells.

Interleukin 17D (IL-17D), a pro-inflammatory cytokine, is a signature cytokine of T helper 17 (Th17) cells. However, studies characterizing the functions of IL-17D in teleost are scarce. Therefore, we aimed to characterize the properties of IL-17D in Amphiprion clarkii. We performed spatial and temporal expression, AcIL-17D-mediated antibacterial and inflammatory gene expression, NFκB pathway-related gene expression analyses, and bacterial colony counting and cell protection assays. We found that AcIL-17D contains a 630 bp coding sequence and encodes 210 amino acids. The spatial expression analysis of AcIL-17D in 12 tissues showed ubiquitous expression, with the highest expression in the brain, followed by blood and skin. Temporal expression analysis of AcIL-17D in blood showed upregulated expression at 6 and 24 h (polyinosinic: polycytidylic acid and lipopolysaccharide), 12 h (all stimulants), and 48 h (polyinosinic: polycytidylic acid and Vibrio harveyi). AcIL-17D expression in the blood gradually decreased at later hours in response to all the stimulants. After treatment of fathead minnow (FHM) cells with different recombinant AcIL-17D concentrations, the downstream gene expression analysis showed increased expression of antimicrobial genes in the FHM cells, namely [NK-Lysin (NKL), Hepcidin antimicrobial peptide-1 (HAMP-1), Defensin-ß (DEFB1)] and some inflammatory genes such as IL-1ß, TNF-α, IL-11, and STAT3. Further nuclear factor κB (NFκB) subunits (NFκB1, NFκB2, RelA, and Rel-B) showed upregulated gene expression at 12 and 24 h. The bacterial colony counting assay using FHM cells showed lower bacterial colony counts in rAcIL-17D-treated cells than in control. Furthermore, the Water-Soluble Tetrazolium Salt (WST -1) assay confirmed the ability of rAcIL-17D in the protection of FHM cells from bacterial infection and conducted the Hoechst 33342 staining upon treatment with rAcIL-17D and rMBP. Therefore, our findings provide important insights into the activation of IL-17D pathway genes in FHM cells, the protective role of AcIL-17D against bacterial infection, and host defense mechanisms in teleost.

Paenihalocynthiibacter styelae gen. nov., sp. nov., isolated from stalked sea squirt Styela clava.

A Gram-stain-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, MYP1-1T, was isolated from the intestine of a stalked sea squirt (Styela clava) of the South Sea in the Republic of Korea. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain MYP1-1T clustered with the type strains of Halocynthiibacter species and Pseudohalocynthiibacter aestuariivivens. Strain MYP1-1T exhibited 16S rRNA gene sequence similarity values of 97.0-97.6 % to the type strains of Halocynthiibacter namhaensis, Halocynthiibacter arcticus and P. aestuariivivens. The phylogenetic tree based on genomic sequences showed that strain MYP1-1T formed a distinct branch separating it from the type strains of two Halocynthiibacter species and P. aestuariivivens and other taxa. The DNA G+C content of strain MYP1-1T from its genomic sequence was 55.0 mol%. Strain MYP1-1T contained Q-10 as the predominant ubiquinone and C18 : 1 ω7c as the major fatty acid. The major polar lipids of strain MYP1-1T were phosphatidylcholine, phosphatidylglycerol, one unidentified lipid and one unidentified aminolipid. The differences in fatty acid and polar lipid profiles and other differential phenotypic properties made it reasonable to distinguish strain MYP1-1T from the genera Halocynthiibacter and Pseudohalocynthiibacter. On the basis of the polyphasic taxonomic investigations, we conclude that strain MYP1-1T constitutes a new genus and species within the class Alphaproteobacteria, for which the name Paenihalocynthiibacter styelae gen. nov., sp. nov. is proposed. The type strain is MYP1-1T (=KCTC 82143T=NBRC 114355T).

Population genetic structure of Semisulcospira gottschei: simultaneous examination of mtDNA and microsatellite markers.

Semisulcospira gottschei is an Asian endemic species inhabiting Korea and China. However, genetic structure analysis of the resource management of this species has not been performed. To investigate the genetic diversity among populations, microsatellites can be used to determine the geographic origins of marine and freshwater species. This study investigated the genetic structures of the Korean and Chinese populations of S. gottschei based on mitochondrial DNA (mtDNA) Cytochrome oxidase subunit I (COI) and polymorphic microsatellite loci developed from Semisulcospira coreana. Analysis of the mtDNA COI sequence revealed 43 haplotypes, which indicated no gene flow between the Korean and Chinese populations. To further elucidate the genetic structures of the Korean and Chinese populations, the population genetics of S. gottschei were analyzed using nine microsatellite markers. The genetic diversity analysis showed an average of 5.25 alleles per locus, with an average allelic richness of 4.02. Excessive homozygosity was found at all loci, which was expected to be due to the presence of null alleles at all loci. Populations of S. gottschei formed two separate clusters according to pairwise FST and AMOVA. Also, the UPGMA tree, PCA, STRUCTURE, and GeneClass indicated separation of the 11 populations into two clusters: Korea and China. These results have potential use in the management, restoration, and distinction of the origin country of populations.

Complement factor D homolog involved in the alternative complement pathway of rock bream (Oplegnathus fasciatus): Molecular and functional characterization and immune responsive mRNA expression analysis.

The complement system serves conventional role in the innate defense against common invading pathogens. Complement factor D (CfD) is vital to alternative complement pathway activation in cleaving complement factor B. This catalytic reaction forms the alternative C3 convertase that is crucial for complement-mediated pathogenesis. In this study, rock bream (Oplegnathus fasciatus) CfD (OfCfD) was characterized and OfCfD mRNA expression was investigated. OfCfD encodes 277 amino acids (aa) for a 30-kDa polypeptide. A domain analysis of the deduced OfCfD aa sequence showed a single serine protease trypsin superfamily domain, a serine active region, three active sites, and three substrate-binding sites. Pairwise sequence comparisons indicated that OfCfD has the highest identity (84.5%) with Oreochromis niloticus CfD. The phylogenetic tree revealed a common ancestral origin of CfD members, with fish CfD distinct from other vertebrate orthologs. The structural arrangement of the OfCfD gene (2451 bp) contained five exons interrupted by four introns. A spatial transcriptional analysis indicated that OfCfD transcripts constitutively expressed in all of the examined rock bream tissues, and that they were highest in the spleen and liver. In addition, OfCfD transcripts were immunologically upregulated by lipopolysaccharide (LPS) (12 h p.i.), Streptococcus iniae (12 h p.i.), rock bream iridovirus (RBIV) (6-12 h p.i.), and poly I:C (6 h p.i.) in spleen tissue. OfCfD is a trypsin protease and its recombinant protein showed strong protease activity similar to that of trypsin, indicating its catalytic function in the alternative pathway. Together, our findings suggest that OfCfD might be involved in immune responses in rock bream.

Thioredoxin domain-containing protein 12 from Oplegnathus fasciatus: Molecular characterization, expression against immune stimuli, and biological activities related to oxidative stress.

Thioredoxin domain-containing protein 12 (TXNDC12) is a small, disulfide-containing protein that belongs to the thioredoxin (TXN) superfamily. In the present study, we identified and characterized a TXNDC12-like gene, designated OfTXNDC12, from rock bream, Oplegnathus fasciatus. OfTXNDC12 consists of seven exons interrupted by six introns. Comparative genomic structural analysis revealed that the TXNDC12 of vertebrates is a structurally conserved gene. The coding sequence of OfTXNDC12 comprises 522 bp, which encodes 173 amino acid residues with the conserved thioredoxin active site motif, CGAC, and a probable C-terminal ER retrieval motif, GDEL. Transcriptional analysis of OfTXNDC12 showed the highest concentrations of the mRNA transcript in the liver, implying that it has a significant role in the liver under normal physiological conditions. In comparison, injection of lipopolysaccharide, Edwardsiella tarda, Streptococcus iniae, polyinosinic:polycytidylic acid (poly[I:C]) and rock bream iridovirus mostly triggered greater upregulation of OfTXNDC12 transcript levels in liver than in gill tissue, supporting its potential functional importance in the liver. Insulin disulfide reduction assay showed that the recombinant fusion protein (rOfTXNDC12) possesses significant thioredoxin activity. Treatment of LNCaP cells with the recombinant protein along with H2O2 revealed that rOfTXNDC12 increased the viability of cells and further supported its putative antioxidant capacity. Taken together, the results from our study suggest that OfTXNDC12 encodes for a potent antioxidant involved in redox regulation that shows significant responses to immune stimuli.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus).

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38ß (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38ß (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (∼ 15.6 kb) and OfMAPK14 (∼ 13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.

A CXC chemokine gene, CXCL12, from rock bream, Oplegnathus fasciatus: Molecular characterization and transcriptional profile.

Chemokines are small, structurally related chemotactic cytokines characterized by the presence of conserved cysteine residues. In the present study, we identified the cDNA of a CXC chemokine from Oplegnathus fasciatus, designated as OfCXCL12. An open reading frame of 297 bp encoded a 98 amino acid peptide with a putative signal peptide of 23 amino acids. The CXC family-specific small cytokine domain (SCY), which is highly conserved among vertebrates, was located between residues 29 and 87. The characteristic conserved cysteine residues in the CXC motif of OfCXCL12 were separated by tyrosine (Y). Similar to other vertebrate CXCL12 proteins, OfCXCL12 also lacked the ELR motif and hence belongs to ELR(-) subfamily. Phylogenetic analysis revealed two distinct clades, consisting of fish and tetrapod CXCL12 homologs. Constitutive expression with significantly higher levels of OfCXCL12 mRNA transcription was detected in immune-related organs, including the head kidney, spleen, and kidney. Infection with bacterial and viral agents led to significant upregulation of mRNA expression in both the head kidney and spleen, in a stimulant-specific manner. Stimulation of peripheral blood leukocytes by the mitogen concanavalin-A significantly induced OfCXCL12 transcription. Results from the present study suggest an important role for OfCXCL12 in immune defense against bacterial and viral infection in rock bream.

Characterization of rock bream (Oplegnathus fasciatus) complement components C1r and C1s in terms of molecular aspects, genomic modulation, and immune responsive transcriptional profiles following bacterial and viral pathogen exposure.

The complement components C1r and C1s play a crucial role in innate immunity via activation of the classical complement cascade system. As initiators of the pathogen-induced signaling cascade, C1r and C1s modulate innate immunity. In order to understand the immune responses of teleost C1r and C1s, Oplegnathus fasciatus C1r and C1s genes (OfC1r and OfC1s) were identified and characterized. The genomic sequence of OfC1r was enclosed with thirteen exons that represented a putative peptide with 704 amino acids (aa), whereas eleven exons of OfC1s represented a 691 aa polypeptide. In addition, genomic analysis revealed that both OfC1r and OfC1s were located on a single chromosome. These putative polypeptides were composed of two CUB domains, an EGF domain, two CCP domains, and a catalytically active serine protease domain. Phylogenetic analysis of C1r and C1s showed that OfC1r and OfC1s were evolutionary close to the orthologs of Pundamilia nyererei (identity = 73.4%) and Oryzias latipes (identity = 58.0%), respectively. Based on the results of quantitative real-time qPCR analysis, OfC1r and OfC1s transcripts were detected in all the eleven different tissues, with higher levels of OfC1r in blood and OfC1s in liver. The putative roles of OfC1r and OfC1s in response to pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus, RBIV) were investigated in liver and head kidney tissues. The transcription of OfC1r and OfC1s was found to be significantly upregulated in response to pathogenic bacterial and viral infections. Overall findings of the present study demonstrate the potential immune responses of OfC1r and OfC1s against invading microbial pathogens and the activation of classical signaling cascade in rock bream.

Mitochondrial peroxiredoxin 3 (Prx3) from rock bream (Oplegnathus fasciatus): immune responses and role of recombinant Prx3 in protecting cells from hydrogen peroxide induced oxidative stress.

Pathogenic infections and environmental factors cause a variety of stresses in fish including oxidative stress by rapid elevation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Transcriptional activation and expression of antioxidant enzymes are essential for reducing the oxidative stress. In this study, we present the molecular characterization, immune responses and ROS scavenging activity of mitochondrial peroxiredoxin 3 from Oplegnathus fasciatus (RbPrx3). Coding sequence (CDS) of RbPrx3 contains 248 amino acids polypeptide which consists of highly conserved peroxiredoxin super family domain and two cysteine residues. Pairwise sequence comparison revealed that RbPrx3 has the greatest identity (94.8%) to Sparus aurata Prx3. Transcriptional analysis of RbPrx3 indicated the ubiquitously expressed mRNA in wide array of organs showing the highest expression in the liver of rock bream. Upon immune challenge of Edwardsiella tarda, Streptococcus iniae, rock bream iridovirus (RBIV) and lipopolysaccharide (LPS), RbPrx3 mRNA level was up-regulated in immunocompetent liver tissues compared to unchallenged fish. Purified recombinant RbPrx3 treated THP-1 cells showed higher survival rate against H(2)O(2) induced oxidative stress and significantly reduced the level of intracellular ROS. Overall results from our study suggest that RbPrx3 may be involved in broader functions such as regulating oxidative stresses by scavenging ROS and activating immune responses in rock bream.

A homolog of Kunitz-type serine protease inhibitor from rock bream, Oplegnathus fasciatus: Molecular insights and transcriptional modulation in response to microbial and PAMP stimulation, and tissue injury.

Serine proteases and their inhibitors play vital roles in diverse biological processes. In this study, we identified and characterized cDNA coding for a Kunitz-type serine protease inhibitor (SPI), which we designated as RbKSPI, in a commercially important species, rock bream. The full-length cDNA sequence of RbKSPI consisted of 2452 bp with an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acid (aa) residues. In the RbKSPI protein, MANEC, PKD, LDLa, and two Kunitz domains responsible for various functions were identified as characteristic features. Homology analysis revealed that RbKSPI shared the highest identity with the Kunitz homolog in Takifugu rubripes (77.6%). Phylogenetic analysis indicated that RbKSPI clusters with other teleostean KSPIs. In tissue-specific expression analysis, RbKSPI transcripts were detected in all the tested tissues, with the highest expression in gill tissue, followed by kidney and intestine. The mRNA expression of RbKSPI significantly increased in blood cells upon stimulation with two strains of bacteria (Edwardsiella tarda and Streptococcus iniae) and two pathogen-associated molecular patterns (PAMPs; LPS and poly I:C). Meanwhile, down-regulated expression of RbKSPI was observed in response to tissue injury. Collectively, these results suggest that the RbKSPI may be involved in essential immune defense against microbial pathogens and in the wound-healing process.

Molecular aspects, genomic arrangement and immune responsive mRNA expression profiles of two CXC chemokine receptor homologs (CXCR1 and CXCR2) from rock bream, Oplegnathus fasciatus.

The CXCR1 and CXCR2 are the prototypical receptors and are the only known receptors for mammalian ELR+ (Glu-Leu-Arg) CXC chemokines, including CXCL8 (interleukin 8). These receptors transduce the ELR+ chemokine signals and operate the downstream signaling pathways in inflammation and innate immunity. In this study, we report the identification and characterization of CXCR1 and CXCR2 genes from rock bream fish (OfCXCR1 and OfCXCR2) at the molecular level. The cDNA and genomic DNA sequences of the OfCXCR1 and OfCXCR2 were identified from a transcriptome library and a custom-constructed BAC library, respectively. Both OfCXCR genes consisted of two exons, separated by an intron. The 5'-flanking regions of OfCXCR genes possessed multiple putative transcription factor binding sites related to immune response. The coding sequences of OfCXCR1 and OfCXCR2 encoded putative peptides of 355 and 360 amino acids (aa), respectively. The deduced aa sequences of OfCXCR1 and OfCXCR2 comprised of a G-protein coupled receptors (GPCR) family 1 profile with a GPCR signature and a DRY motif. In addition, seven conserved transmembrane regions were predicted in both OfCXCRs. While our multiple alignment study revealed the functionally significant conserved elements of the OfCXCR1 and OfCXCR2, phylogeny analyses further confirmed their position in teleost sub clade, in which they manifested an evolutionary relatedness with other fish counterparts. Based on comparative analyses, teleost CXC chemokine receptors appear to be distinct from their non-fish orthologs in terms of evolution (both CXCR1 and CXCR2) and genomic organization (CXCR2). Quantitative real-time PCR (qPCR) detected the transcripts of OfCXCR1 and OfCXCR2 in eleven examined tissues, with higher levels in head kidney, kidney and spleen highlighting their crucial importance in immunity. In vitro stimulation of peripheral blood leukocytes (PBLs) with concanavalin A (Con A) resulted in modulation of OfCXCR2 transcription, but not that of OfCXCR1. In addition, the magnitude of the OfCXCR1 and OfCXCR2 transcripts in head kidney and spleen was differentially increased after the in vivo administration of immune stimulants, LPS and poly I:C and in the infection models injected with rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. These lines of evidence suggest that these receptors may play an important role(s) in immune responsive signaling during pathogenesis of rock bream.

Two NF-κB inhibitor-alpha (IκBα) genes from rock bream (Oplegnathus fasciatus): molecular characterization, genomic organization and mRNA expression analysis after immune stimulation.

IkBa is a member of IkB family, which sequesters NF-kB in an inactivate form in the cytoplasm and blocks the translocation of NF-kB to nucleus. The IkBa paralogs of rock bream (OfIkBa-A and OfIkBa-B) encoded IkBa proteins with typical features including, highly conserved IkB degradation motif, six ankyrin repeats and a PEST sequence. However, their amino acid identity and similarity were only 55.6 and 69.7%, respectively suggesting that these two genes could be the two different isoforms of IkBa. The number and size of the exons of OfIkBa-A and OfIkBa-B were conserved well with all the compared vertebrate species, although they have significantly different genomic sizes. Phylogenetic analysis revealed that OfIkBa-A and OfIkBa-B proteins cluster with IkBa family members; however, they were grouped with different subclades in IkBa family. Tissue specific expression of OfIkBa mRNA was constitutively detected in all the tested tissues, and they showed the higher transcription level in heart, liver, gill and peripheral blood cells, respectively. The injection of flagellin stimulated the mRNA expression of OfIkBa paralogs in head kidney and intestine. Moreover, the OfIkBa mRNA expression in gill and liver was significantly upregulated by LPS, poly I:C and Edwardsiella tarda challenges. The transcription of OfIkBa was up-regulated in early-phase of injection and then rapidly restored. These results suggest that the OfIkBa paralogs might be involved in rapid immune responsive reactions in rock bream against bacterial and viral pathogens.

A teleostean angiotensinogen from Oplegnathus fasciatus responses to immune and injury challenges.

Angiotensinogen (AGT) is the precursor of the renin-angiotensin system and contributes to osmoregulation, acute-phase and immune responses. A full-length cDNA of the AGT (2004 bp with a 1389 bp coding region) was isolated from rock bream (Rb), Oplegnathus fasciatus. The encoded polypeptide of 463 amino acids had a predicted molecular mass of 51.6 kDa. RbAGT possessed a deduced signal peptide of 22 residues upstream of a putative angiotensin I sequence ((23)NRVYVHPFHL(32)). RbAGT possessed a specific domain profile and a signature motif which are characteristics of the serpin family. Sequence homology and phylogenetic analysis indicated that RbAGT was evolutionarily closest to AGT of Rhabdosargus sarba. The mRNA expression profile of RbAGT was determined by quantitative RT-PCR and it demonstrated a constitutive and tissue-specific expression with the highest transcript level in the liver. Significantly up-regulated RbAGT expression was elicited by systemic injection of a lipopolysaccharide, rock bream iridovirus (RBIV) and bacteria (Edwardsiella tarda and Streptococcus iniae), revealing its pathogen inducibility. RbAGT manifested a down-regulated response to systemic injury, contemporaneously with two other serpins, protease nexin-1 (PN-1), and heparin cofactor II (HCII). In addition, a synchronized expression pattern was elicited by RbAGT and RbTNF-α in response to injury, suggesting that TNF-α might be a potential modulator of AGT transcription.

The complete mitochondrial genome and phylogenetic position of the Scalpel sawtail Prionurus scalprum Valenciennes, 1835 (Acanthuriformes: Acanthuridae) from Jeju Island, Korea.

Although the mitochondrial genomes of most Acanthuriformes fish have been sequenced, this has not been done for Prionurinae fish yet. The Scalpel sawtail (Prionurus scalprum Valenciennes, 1835) found in all tropical and sub-tropical seas, is an important link between primary producers and higher trophic levels. In this study, the complete mitochondrial genome of the Scalpel sawtail, Prionurus scalprum Valenciennes, 1835 (accession number: OK323243) was sequenced. The complete mitochondrial genome included 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region; the total length was 16,522 bp. The nucleotide composition of the genome was as follows: A, 28.46%; T, 27.62%; G, 16.46%; and C, 27.46%. All genes were encoded on the H-strand, except for the eight tRNA genes and NADH dehydrogenase subunit (ND6). A phylogenetic tree was constructed using the neighbor-joining (NJ) method and the phylogenetic position of the Scalpel sawtail was established. This provides useful data for future research on Acanthuridae fish.

Genotyping-by-Sequencing of the regional Pacific abalone (Haliotis discus) genomes reveals population structures and patterns of gene flow.

Continuous monitoring of the present genetic status is essential to preserve the genetic resource of wild populations. In this study, we sequenced regional Pacific abalone Haliotis discus samples from three different locations around the Korean peninsula to assess population structure, utilizing Genotyping-by-Sequencing (GBS) method. Using PstI enzyme for genome reduction, we demonstrated the resultant library represented the whole genome region with even spacing, and as a result 16,603 single nucleotide variants (SNVs) were produced. Genetic diversity and population structure were investigated using several methods, and a strong genetic heterogeneity was observed in the Korean abalone populations. Additionally, by comparison of the variant sets among population groups, we were able to discover 26 Korean abalone population-specific SNVs, potentially associated with phenotype differences. This is the first study demonstrating the feasibility of GBS for population genetic study on H. discus. Our results will provide valuable data for the genetic conservation and management of wild abalone populations in Korea and help future GBS studies on the marine mollusks.

Classification of Takifugu rubripes, T. chinensis and T. pseudommus by genotyping-by-sequencing.

Takifugu rubripes is more expensive than other species of the genus because of its high protein content and special flavor. However, it is easily confused with imported T. chinensis and T. pseudommus because they have similar morphological characteristics. We identified single nucleotide polymorphism (SNP) markers of T. rubripes by genotyping-by-sequencing (GBS) and evaluated their ability to distinguish among T. rubripes, T. chinensis, and T. pseudommus. In all, 18 polymorphic SNPs were subjected to phylogenetic analyses of the three Takifugu species. Additionally, we subjected a second set of samples to Sanger sequencing to verify that the polymorphic SNPs could be used to evaluate the genetic variation among the three Takifugu species. A phylogenetic tree that included the analyzed sequence of set A, which is referred to as the reference sequence, and a validation sequence of set B with 18 SNPs were produced. Based on this phylogenetic tree and STRUCTURE analyses, T. rubripes, T. chinensis and T. pseudommus have low genetic variation and should be considered the same gene pool. Our findings suggest that further studies are needed to estimate the genetic association of the three Takifugu species.

Molecular cloning and characterization of olive flounder (Paralichthys olivaceus) peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that play key roles in lipid and energy homeostasis. Olive flounder (Paralichthys olivaceus) PPARgamma cDNA (olPPARgamma) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full-length cDNA is 1667-bp long and encodes a polypeptide with 532 amino acids containing a C4-type zinc finger and a ligand-binding domain. Quantitative RT-PCR revealed that olPPARgamma transcription was detected from 7days post-hatching, and its expression was increased under a starved condition. Overexpression of olPPARgamma stimulated PPAR response element (PPRE) activity, and treatment with rosiglitazone, a PPARgamma agonist, augmented olPPARgamma-stimulated PPRE activity in HINAE olive flounder cells. Cotransfection of olPPARgamma and olRXRbeta, in the absence or presence of rosiglitazone and ciglitazone, produced a synergistic effect on PPRE transactivation in 3T3L1 adipocytes. Moreover, olPPARgamma, in the presence or absence of rosiglitazone, regulated the expression of lipid synthesis- and adipogenesis-related proteins in NIH3T3 and 3T3L1 cells. Taken together, these results suggest that olPPARgamma is functionally and evolutionarily conserved in olive flounder and mammals.

The Regulatory Region of Muscle-Specific Alpha Actin 1 Drives Fluorescent Protein Expression in Olive Flounder Paralichthys olivaceus.

To develop a promoter capable of driving transgene expression in non-model fish, we identified and characterized the muscle-specific alpha-actin gene in olive flounder, Paralichthys olivaceus (PoACTC1). The regulatory region of PoACTC1 includes putative regulatory elements such as a TATA box, two MyoD binding sites, three CArG boxes, and a CCAAT box. Microinjection experiments demonstrated that the regulatory region of PoACTC1, covering from -2,126 bp to +751 bp, just prior to the start codon, drove the expression of red fluorescent protein in developing zebrafish embryos and hatching olive flounder. These results suggest that the regulatory region of PoACTC1 may be useful in developing a promoter for biotechnological applications such as transgene expression in olive flounder.

Development of Species-Specific PCR Primers for the Rapid and Simultaneous Identification of the Six Species of Genus Takifugu.

Pufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

Segregation distortion at a microsatellite marker in the olive flounder, Paralichtys olivaceus.

In the course of quantitative trait loci (QTL) analysis of the back cross (BC1) families of olive flounder (Paralichtys olivaceus), we observed significant segregation distortion at a microsatellite marker Poli9-58TUF in two crosses of informative progenies. The family 1 of the random BC1 progenies derived from a cross between a F1 male genotype (A/B) and a F1's female parent genotype (A/C) and the family 2 (A/C x A/C) displayed a strong bias in the locus from the Mendelian inheritance by the elimination of homozygous A/A genotype. The deleterious roles of the AA genotypes are suggested during the metamorphosis and it implies that the parents of these families carried a recessive gene or genes hampering development at an early stage because the offspring of the double heterozygote parents show the reduction in frequency or elimination of one homozygous class, which is an evidence for linkage between the genetic marker and gene(s) with recessive deleterious alleles. This data support a hypothesis that the region contains a recessive lethal gene or genes.