The study on the complete mitochondrial genome of Acanthopsetta nadeshnyi and its phylogenetic position.

Pleuronectidae is a well-studied familyin the order Pleuronectiformes. In contrast, genetic research on the flatfish Acanthopsetta nadeshnyi of the Pleuronectidae family is limited. This study reports the complete mitogenome of A. nadeshnyi. The mitogenome was 17,206 bases long and included 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a putative control region. Phylogenetic analysis based on the nucleotide sequences of the 13 PCGs confirmed that A. nadeshnyi belongs to the Pleuronectidae family.

Characteristics and phylogenetic analysis of the complete mitochondrial genome of Microstomus achne.

Microstomus achne (Jordan and Starks, 1904) is an economically valuable flatfish belonging to the family Pleuronectidae and the only flatfish that inhabits Korea. Here, we report on the complete mitochondrial genome of M. achne and the phylogenetic relationship between close species. The mitogenome is 16,971 bp long and encodes 13 protein-coding genes (PCGs), 22 transfer RNAs, and two ribosomal RNAs. The phylogenetic analysis showed that M. achne clustered with Glyptocephalus stelleri, which supports the conclusion that M. achne belongs to the family Pleuronectidae. The results of this study provide a better understanding of M. achne.

Characterization and phylogenetic analysis of the complete mitochondrial genome of Notostomum cyclostomum.

Notostomum cyclostomum is a parasite that lays eggs on the snow crab shells and causes various diseases by parasitizing fish. Although there have been some studies on the life history of this parasite and the associated fish diseases, little is known about the molecular biology of this parasite. Thus, here we report the mitochondrial genome of N. cyclostomum, which is 16,972 bp long and contains 13 mitochondrial protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a putative control region with a 92% AT-rich sequences between tRNA-R and tRNA-H. Phylogenetic analysis using 13 PCGs confirmed that N. cyclostoum belongs to the family Pisciodlidae. This is the first study revealing the complete mitochondrial genome sequence of N. cyclostomum.

The complete mitochondrial genome of Pseudopleuronectes herzensteini.

Pseudopleuronectes herzensteini belonging to Pleuronectiformes (family Pleuronectidae) is important in the fishery industry. However, the molecular biology of this valuable fish has hardly been reported. Thus, here we report the complete mitochondrial genome of P. herzensteini. The mitochondrial DNA (mtDNA) of P. herzensteini is 16,719 bp long and contains 13 mitochondrial protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a putative control region between tRNA-P and tRNA-F distinguished by a single short noncoding region. Phylogenetic analysis using PCGs confirmed that this mtDNA sequence belongs to the family Pleuronectidae. This is the first study reporting the complete mitochondrial genome sequence of P. herzensteini.

Complete mitochondrial DNA sequence of Norwegian skates (Raja brachyuran Lafont, 1871) imported to Korea.

In this study, the complete mitochondrial genome of Norwegian skates imported to Korea was sequenced with a circular molecule of 17,121 bp, which consisted of 13 protein-coding genes (PCGs), 2 ribosomal RNAs, 22 transfer RNA genes, and a control region (D-loop). And among these sequences, 193 bp sequence in the D-loop of the genus Raja suggested the possibility of being used as a genetic marker for classification of Raja and Dipturus species. The BI phylogenetic tree by using the nucleotide sequences of 13 PCGs from 15 available mitogenomes of family Rajidae confirmed also that Norwegian skates imported to Korea form a group with Raja brachyura species with high branch value, and that this was a species of Raja brachyura. As above, these results would be expected to provide for the further understanding on the phylogenetic relationship, taxonomic classification and phylogeography of the family Rajidae.

The complete mitochondrial genome of Sardinella zunasi (Clupeiformes: Clupeidae).

The complete mitogenome of Sardinella zunasi was determined by next-generation sequencing. The S. zunasi mitogenome was a circular 16,307 bp molecule that contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and one control region (D-loop). The gene arrangement was consistent with other Sardinella mitogenomes. The phylogenetic relationships of 29 Clupeoidei species based on 13 protein-coding genes from the available mitogenomes were analyzed. Sardinella zunasi clustered with Sardinella among Clupeidae, suggesting a closer relationship with this genus. These results will be useful for understanding the phylogenetic relationships, taxonomic classification, and phylogeography of the genus Sardinella relative to other genera of Clupeoidei.

The complete mitochondrial genome of an edible mushroom, Sparassis crispa.

Sparassis crispa, also known as cauliflower mushroom, is a widely used medicinal mushroom in traditional Chinese medicine due to the presence of bioactive substances with pharmacological activity. Here, we report a complete mitochondrial genome sequence of S. crispa consisting of 139,253 bp containing 47 genes including 15 protein-coding genes, 27 transfer RNA, and 5 ribosomal RNA genes obtained from 40.406 Mb genome containing 18,917 predicted contigs using raw data of next-generation sequencing having 85.4% Q30. The overall base composition of S. crispa was 26.47% G-C and 73.53% A-T. The phylogenetic tree based on atp6 sequence data showed its close relationship with Sparassis radicata. The complete mitochondrial genome sequence of S. crispa provides an essential and important DNA molecular data for further phylogenetic and evolutionary analysis of S. crispa.

Complete mitochondrial genome of Halocynthia hilgendorfi ritteri (Pyuridae) from Korea.

Halocynthia hilgendorfi ritteri is an ascidian distributed on the coast of Geoje Island in Korea and found on rocks. The mitochondrial genome of Halocynthia hilgendorfi ritteri consists of 15,181 bp with 13 protein-coding genes, 2 ribosomal RNAs, 23 transfer RNA genes. The overall base composition of the complete genome is 22.94% A, 43.32% T, 25.72% G, and 8.02% C, with a high A + T content of 66.26%.

The complete mitochondrial genome analysis and phylogenetic position of the short barbeled grunter Hapalogenys nigripinnis (Lobotiformes: Hapalogenyidae) from Jeju island, Korea.

This study analyzed the complete mitochondrial genome of the short barbeled grunter Hapalogenys nigripinnis (Accession number: MT374064). The complete mitogenome was 16,476 bp long and included 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region. Nucleotide composition of the genome was A: 28.70%, T:27.46%, G: 15.73%, and C: 28.11%. All genes were encoded on the H-strand, except for the NADH dehydrogenase subunit (ND6) and 8 tRNA genes. When compared this sequence with the mitogenome of Chinese black grunt, Korean short barbeled grunter showed difference of 64 bp of nucleotide sequence in 20 genes. Phylogenetic tree was constructed using the neighbor-joining (NJ) method and showed the phylogenetic position of the short barbeled grunter in Korea.

Genetic diversity and population structure of the endangered fish Pseudobagrus brevicorpus (Bagridae) using a newly developed 12-microsatellite marker.

BACKGROUND: Pseudobagrus brevicorpus is an endangered species in Korea. The development of genetic markers and genetic information regarding the populations of this species are needed to establish conservation strategies. OBJECTIVE: As part of the conservation of P. brevicorpus, a 12-microsatellite marker was developed using next-generation sequencing (NGS) to provide current genetic population information. METHODS: Microsatellites from P. brevicorpus were identified by NGS analysis. Genetic diversity and genetic structure analysis of six populations (Seojeong Stream [SJ], Gokgang Stream [GK], Jaho Stream [JH], Daega Stream [DG], Nam River [NG], and Deokcheon River [DC]) of P. brevicorpus were conducted using the newly developed microsatellite marker. RESULTS: NGS generated 10,347,578 reads and identified 659,507 simple sequence repeats. Twelve microsatellites were successfully amplified and verified in 30 individuals of P. brevicorpus. The genetic diversity of the six P. brevicorpus populations in terms of the number of alleles ranged from 3.667 to 7.111. All populations except DG deviated significantly from Hardy-Weinberg equilibrium (HWE) at one or more loci. The genetic distances of the six populations showed the closest relationship between the SJ and GK populations (independent Stream populations), and there was a close relationship with the JH population among the Nakdong River. Structure analysis showed that P. brevicorpus is largely divided into two groups. CONCLUSIONS: The developed microsatellite marker will be used to provide basic genetic data of P. brevicorpus. Genetic diversity and structure analysis of the population will provide useful information for conservation management of P. brevicorpus.

The complete mitochondrial genome sequence and phylogenetic analysis for Inimicus japonicus (Scorpaeniformes: Synanceiidae).

The complete mitochondrial genome (mitogenome) of the Inimicus japonicus was analyzed by next-generation sequencing in this study. The mitogenome is 16,978 base pairs (bp) long and codes for 22 transfer RNA (tRNA) genes, 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNA), and 1 non-coding control region. The overall nucleotide composition of the mitogenome is: 29.61% for A, 29.16% for T, 25.26% for C, and 15.97% for G. Twenty-two tRNAs range from 67 to 74 bp in length, and 2 rRNA (12S and 16S) were 953 and 1,687 bp long, respectively. Phylogenetic analysis by neighbor-joining (NJ) method indicated that I. japonicus showed considerable genetic similarity (82%), and had a closer relationship in the phylogenetic tree to Synanceia verrucosa.

The complete mitochondrial genome of Chionoecetes japonicus (Crustacea: Decapoda: Majoidea).

The complete mitochondrial genome of Chionoecetes japonicus was sequenced using a specimen collected offshore in the East Sea. The genome includes 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (D-loop), with a total length of 16,060 bp. The overall nucleotide composition was 34.91% A, 17.29% C, 10.93% G, and 36.87% T, with 71.78% A + T. In the phylogenetic tree was constructed using maximum-likelihood and Bayesian inference analyses, C. japonicus and C. japonicus pacificus formed a genetic clade that was sister to C. opilio.

Antidiabetic and antioxidant activities of red seaweed Laurencia dendroidea

Objective: To investigate antidiabetic and antioxidant activities of the extract and fractions from Vietnamese red seaweed Laurencia dendroidea. Methods: The seaweed Laurencia dendroidea was extracted by using microwave-assisted extraction method in 80％ methanol. The seaweed extract was then fractionated using different solvents (n-hexane, chloroform, ethyl acetate, butanol and water). These obtained fractions were evaluated for α-glucosidase inhibitory and antioxidant activities. Antioxidant activities were tested using DPPH, nitric oxide radical scavenging and metal chelating assays. The enzyme inhibition mode was determined using Lineweaver-Burk plot. For acidic and thermal stabilities, the ethyl acetate fraction was treated at pH 2.0 and 100 ℃, respectively. The residual inhibitory activity of the fraction was calculated based on the initial inhibitory activity. For in vivo antidiabetic activity, mice were divided into four groups, including normal control, diabetic control, diabetic mice treated with ethyl acetate fraction and diabetic mice treated with gliclazide. Blood glucose level of treated mice during acute and prolonged treatments was measured. To evaluate the toxicity of the ethyl acetate fraction, the body weight changes and activities of liver function enzymes (aspartate transaminase, alanine transaminase and gammaglutamyl transferase) were carried out. Results: The extract of Laurencia dendroidea showed strong α-glucosidase inhibitory and DPPH radical scavenging activities. Methanolic concentrations affected both α-glucosidase inhibitory and antioxidant activities. A 80％ aqueous methanol was the suitable solvent for extraction of enzyme inhibitors and antioxidants. Among solvent fractions, ethyl acetate fraction had the highest inhibitory activities against α-glucosidase with a mixed type of inhibition and the strongest antioxidant activities, and was stable under acidic and thermal conditions. The ethyl acetate fraction treated diabetic mice significantly reduced blood glucose level compared with the diabetic control group (13.16 mmol/L vs. 22.75 mmol/L after 3 hours of treatment). Oral administration of ethyl acetate fraction did not exhibit toxicity at a dose of 100 mg/kg body weight as determined by body weight changes and liver biochemical parameters. Conclusions: Laurencia dendroidea could be a potential source for production of antidiabetic and antioxidative agents.

Bioflocculation of the oceanic microalga Dunaliella salina by the bloom-forming dinoflagellate Heterocapsa circularisquama, and its effect on biodiesel properties of the biomass.

The aim of this work was to evaluate the flocculation by the dinoflagellate Heterocapsa circularisquama as a means for harvesting three Chlorophyta species, Chlorella vulgaris, Nannochloropsis granulata, and Dunaliella salina. Relative fluorescence of D. salina culture significantly decreased along with 9.3-fold increased flocculation activity within 24 h when mixed with H. circularisquama. Lipid content of bioflocculated D. salina increased about 40%, while fatty acid methyl ester (FAME) profiles exhibited higher levels of C16:0, C18:0, and C18:1, compared to harvest by centrifugation, suggesting higher energy content. Furthermore, bioflocculated D. salina biomass had more suitable biodiesel properties relative to both EN14214 and ASTMD6751, with a cetane number of 49.0 and an iodine value of 95.9. These results suggest that H. circularisquama-induced bioflocculation is applicable for the sustainable and qualitative production of algal biodiesel.

Functional analysis of the extended N-terminal region in PLC-δ1 (MlPLC-δ1) from the mud loach, Misgurnus mizolepis.

Mud loach phospholipase C-δ1 (MlPLC-δ1) contains all the characteristic domains found in mammalian PLC-δ isozymes (pleckstrin homology domain, EF-hands, X­Y catalytic region, and C2 domain) as well as an extended 26-amino acid (aa)-long N-terminal region that is an alternative splice form of PLC-δ1 and is novel to vertebrate PLC-δ. In the present structure-function analysis, deletion of the extended N-terminal region caused complete loss of phosphatidylinositol (PI)- and phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity in MlPLC-δ1. Additionally, recombinant full-length MlPLC-δ1 PLC activity was reduced in a dose-dependent manner by coincubation with the 26-aa protein fragment. Using a protein-lipid overlay assay, both full-length MlPLC-δ1 and the 26-aa protein fragment had substantial affinity for PIP2, whereas deletion of the 26-aa region from MlPLC-δ1 (MlPLC-δ1-deletion) resulted in lower affinity for PIP2. These results suggest that the novel N-terminal exon of MlPLC-δ1 could play an important role in the regulation of PLC-δ1.

A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources.

PURPOSE: To evaluate the abilities of these subtyping methods, we distinguished Salmonella Enteritidis (S. Enteritidis) isolated from food products and human clinical samples between 2009 and 2010 in Seoul using five subtyping methods. METHODS: We determined the subtypes of 20 S. Enteritidis isolates from food and human sources using phage typing, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multi-locus sequence typing (MLST). RESULTS: A total of 20 tested isolates were differentiated into six antimicrobial susceptibility patterns, three different phage types, four different PFGE profiles, seven rep-PCR patterns, and one MLST type. Food isolates were considerably more susceptible to antibiotics than human isolates. We were best able to discriminate among S. Enteritidis isolates using rep-PCR, and obtained the highest Simpson's diversity index of 0.82, whereas other methods produced indices that were less than 0.71. PFGE pattern appeared to be more related to antimicrobial resistance and phage types of S. Enteritidis isolates than rep-PCR. MLST revealed identical alleles in all isolates at all seven loci examined, indicating no resolution. CONCLUSION: The results of this study suggest that rep-PCR provided the best discriminatory power for phenotypically similar S. Enteritidis isolates of food and human origins, whereas the discriminatory ability of MLST may be problematic because of the high sequence conservation of the targeted genes.

Expression analysis and enzymatic characterization of phospholipase Cδ4 from olive flounder (Paralichthys olivaceus).

Phospholipase Cδ4 (PLCδ4) plays a significant role in cell proliferation, tumorigenesis, and in an early stage of fertilization. Despite the characterization of the mammalian PLCδ4, extensive study in aquatic organisms has not been carried out so far. Here, we performed the molecular and biochemical characterization of flatfish Paralichthys olivaceus PLCδ4 (PoPLCδ4) to understand its enzymatic properties and physiological functions. The olive flounder PLCδ4 cDNA has an open reading frame (ORF) of 2,268 bp, and encodes a 755 amino acid polypeptide with a predicted molecular weight of 86 kDa. All the characteristic domains found in mammalian PLCδ isoforms (PH domain, EF hands, an X-Y catalytic region, and a C2 domain) were found to be present in PoPLCδ4. The mRNA expression analysis of PoPLCδ4 showed that PoPLCδ4 is predominantly expressed in the brain, eye and heart tissues. Like other mammalian PLCδ proteins, the enzyme activity of recombinant PoPLCδ4 to phosphatidylinositol-4,5-bis-phosphate (PIP2) was noted to be concentration- and Ca(2+)-dependent. The structural features and biochemical characteristics of PoPLCδ4 were found to be similar to those of mammalian PLCδ4. This is the first demonstration of the expression analysis and enzymatic characterization of piscine PLCδ4.

PLC-δ1-Lf, a novel N-terminal extended phospholipase C-δ1.

Phospholipase C-δ (PLC-δ), a key enzyme in phosphoinositide turnover, is involved in a variety of physiological functions. The widely expressed PLC-δ1 isoform is the best characterized and the most well understood phospholipase family member. However, the functional and molecular mechanisms of PLC-δ1 remain obscure. Here, we identified that the N-terminal region of mouse PLC-δ1 gene has two variants, a novel alternative splicing form, named as long form (mPLC-δ1-Lf) and the previously reported short form (mPLC-δ1-Sf), having exon 2 and exon 1, respectively, while both the gene variants share exons 3-16 for RNA transcription. Furthermore, the expression, identification and enzymatic characterization of the two types of PLC-δ1 genes were compared. Expression of mPLC-δ1-Lf was found to be tissue specific, whereas mPLC-δ1-Sf was widely distributed. The recombinant mPLC-δ1-Sf protein exhibited higher activity than recombinant mPLC-δ1-Lf protein. Although, the general catalytic and regulatory properties of mPLC-δ1-Lf are similar to those of PLC-δ1-Sf isozyme, the mPLC-δ1-Lf showed some distinct regulatory properties, such as tissue-specific expression and lipid binding specificity, particularly for phosphatidylserine.

Functional analysis of duplicated genes and N-terminal splice variant of phospholipase C-δ1 in Paralichthys olivaceus.

Phosphoinositide-specific phospholipase C δ (PLC δ) plays an important role in many cellular responses and is involved in the production of second messenger. Here, we describe the presence of novel N-terminal extended alternative splice form of PLC-δ1B in Paralichthys olivaceus, which differs from the reported mammalian PLC-δ1 isoform. The two variants PoPLC-δ1B-Lf and PoPLC-δ1B-Sf share exon 3 (including the PH domain) to exon 16, but differ at the exon 1 (Short form: Sf) and novel exon 2 (Long form: Lf) of the transcript. For the characterization of the novel duplicated gene variant of PLC-δ1B in P. olivaceus, tissue-specific expression with RT-PCR and real-time PCR, and purification and enzymatic characterization of native and recombinant proteins of all the three-types of PLC-δ1 isoforms (PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf) of P. olivaceus were studied. The PoPLC-δ1A was ubiquitously distributed in gill, kidney and spleen. The PoPLC-δ1B-Lf gene was widely detected in various tissues, especially in the digestive system, while PoPLC-δ1B-Sf was highly expressed in the stomach. The recombinant PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins were expressed as a histidine-tagged fusion protein in Escherichia coli. The PLC activity of the PoPLC-δ1 isoform proteins showed a concentration-dependent activity to phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). In addition, U73122, the PLC inhibitor, effectively inhibited PLC activities of PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins. However, PoPLC-δ1A and PoPLC-δ1B-Lf were sensitive at pH 7.5, while PoPLC-δ1B-Sf was relatively sensitive at pH 7. These results might be useful for the study of phospholipase C-mediated signal transduction in fish.

Development of a selective enrichment broth supplemented with bacteriological charcoal and a high concentration of polymyxin B for the detection of Campylobacter jejuni and Campylobacter coli in chicken carcass rinses.

A new Campylobacter-selective enrichment broth supplemented with bacteriological charcoal and a high concentration of polymyxin B was developed (charcoal-cefoperazone-polymyxin B-deoxycholate broth; CCPD broth). We compared the ability of CCPD broth to detect Campylobacter jejuni and Campylobacter coli in chicken carcass rinses to that of modified Bolton (mBolton) broth. Eighty whole chickens were purchased from retailers and rinsed with 400 mL buffered peptone water. The rinsed samples were enriched with 2× blood-free mBolton enrichment broth or 2× CCPD broth at 42 °C for 48 h and then streaked onto modified charcoal-cefoperazone-deoxycholate agar (mCCDA). The Campylobacter isolation rate was significantly higher in CCPD broth than in mBolton broth (CCPD broth, 61 out of 80; mBolton broth, 34 out of 80; p<0.05). Moreover, the selectivity of CCPD broth agar was also superior to that of mBolton broth when comparing the number of contaminated mCCDA plates (CCPD broth, 16 out of 80; mBolton broth, 58 out of 80; p<0.05) and the growth index of competing flora (CCPD broth, 1.4; mBolton broth, 2.9; p<0.05).