Mun Gyeong Kwon

The effects of combined dietary probiotics Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 on innate immunity and disease resistance in olive flounder (Paralichthys olivaceus).

The effects of a dietary probiotic mixture containing Lactococcus (Lc.) lactis BFE920 isolated from bean sprout and autochthonous Lactobacillus (Lb.) plantarum FGL0001 originally isolated from the hindgut of olive flounder (Paralichthys olivaceus) were investigated for the purpose of improving the probiotic effects of Lc. lactis BFE920 on the olive flounder. The immunostimulatory, disease protective, and weight gain effects of Lc. lactis BFE920 were significantly improved when olive flounder (average weight 37.5±1.26 g) were fed the probiotic mixture (log10 7.0 CFU each/g feed pellet) for 30 days. Flounder fed the mixture showed improved skin mucus lysozyme activity and phagocytic activity of innate immune cells compared to flounder fed a single probiotic agent or a control diet. While the levels of neutrophil activity in flounder fed the single probiotic agent or the mixture were similar, they were significantly higher than levels in a control group. Additionally, probiotic-fed flounder showed significantly increased expressions of IL-6, IL-8, and TNF-α in the intestine compared to the control group. Following a 30-day period of being fed probiotics or a control diet, the olive flounder were challenged with an i.p. injection of Streptococcus iniae (log10 6.0 CFU/fish). The groups fed the mixed probiotics, Lc. lactis BFE920, Lb. plantarum FGL0001, and the control diet had survival rates of 55%, 45%, 35%, and 20%, respectively. Flounder fed the probiotic mixture gained 38.1±2.8% more body weight compared to flounder fed the control diet during the 30-day study period. These data strongly suggest that a mixture of Lc. lactis BFE920 and Lb. plantarum FGL0001 may serve as an immunostimulating feed additive useful for disease protection in the fish farming industry.

Non-specific Defensive Factors of the Pacific Oyster Crassostrea gigas against Infection with Marteilioides chungmuensis: A Flow-Cytometric Study

In order to assess changes in the activity of immunecompetency present in Crassostrea gigas infected with Marteilioides chungmuensis (Protozoa), the total hemocyte counts (THC), hemocyte populations, hemocyte viability, and phagocytosis rate were measured in oysters using flow cytometry. THC were increased significantly in oysters infected with M. chungmuensis relative to the healthy appearing oysters (HAO) (P<0.05). Among the total hemocyte composition, granulocyte levels were significantly increased in infected oysters as compared with HAO (P<0.05). In addition, the hyalinocyte was reduced significantly (P<0.05). The hemocyte viability did not differ between infected oysters and HAO. However, the phagocytosis rate was significantly higher in infected oysters relative to HAO (P<0.05). The measurement of alterations in the activity of immunecompetency in oysters, which was conducted via flow cytometry in this study, might be a useful biomarker of the defense system for evaluating the effects of ovarian parasites of C. gigas.

Pathological characteristics of olive flounder Paralichthys olivaceus experimentally infected with Streptococcus parauberis

Pathological characteristics of olive flounder Paralichthys olivaceus experimentally infected with Streptococcus parauberis were studied. Various stressful conditions, aeration and netting stress in particular, led to induced mortality by S. parauberis. Netting stress-induced mortality was positively correlated to bacterial dose and stressful conditions. Inflammation of the heart and pericarditis was the major pathological change observed in olive flounder experimentally infected with S. parauberis. During the infected period, the number of bacteria in the infected olive flounder was recorded over time. S. parauberis remained in all fish organs tested, especially in the heart and brain.

Manganese-superoxide dismutase (MnSOD), a role player in seahorse (Hippocampus abdominalis) antioxidant defense system and adaptive immune system

Abstract Manganese superoxide dismutase (MnSOD) is a metaloenzyme that catalyzes dismutation of the hazardous superoxide radicals into less hazardous H2O2 and H2O. Here, we identified a homolog of MnSOD from big belly seahorse (Hippocampus abdominalis; HaMnSOD) and characterized its structural and functional features. HaMnSOD transcript possessed an open reading frame (ORF) of 672 bp which codes for a peptide of 223 amino acids. Pairwise alignment showed that HaMnSOD shared highest identity with rock bream MnSOD. Results of the phylogenetic analysis of HaMnSOD revealed a close proximity with rock bream MnSOD which was consistent with the result of homology alignment. The intense expression of HaMnSOD was observed in the ovary, followed by the heart and the brain. Further, immune related responses of HaMnSOD towards pathogenic stimulation were observed through bacterial and viral challenges. Highest HaMnSOD expression in response to stimulants Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide (LPS), and polyinosinic‐polycytidylic acid (Poly I:C) was observed in the late stage in the blood tissue. Xanthine/xanthine oxidase assay (XOD assay) indicated the ROS‐scavenging ability of purified recombinant HaMnSOD (rHaMnSOD). The optimum conditions for the SOD activity of rHaMnSOD were pH 9 and the 25 °C. Collectively, the results obtained through the expressional analysis profiles and the functional assays provide insights into potential immune related and antioxidant roles of HaMnSOD in the big belly seahorse. HighlightsMnSOD was identified from big belly seahorse (HaMnSOD).HaMnSOD was cloned and expressed to evaluate its distinct functional features.XOD (Xanthine oxidase) assay confirmed the superoxide scavenging ability of HaMnSOD.Transcriptional level of HaMnSOD was modulated by pathological stress.

Genome Sequence of Streptococcus parauberis Strain KCTC11980, Isolated from Diseased Paralichthys olivaceus

ABSTRACT Streptococcus parauberis is a coccoid, nonmotile, alpha-hemolytic, Gram-positive bacterium of the Streptococcaceae family. Streptococcus parauberis strain KCTC11980 was isolated from the kidney of a diseased olive flounder collected from an aquaculture farm on Jeju Island in 2010. The 2.12-Mb genome sequence consists of 44 large contigs in 16 scaffolds and contains 2,214 predicted protein-coding genes, with a G+C content of 35.4%.

Isolation and characterization of polymorphic microsatellite markers for the black rockfishSebastes inermis

Black rockfish (Sebastes inermis, Scorpaenidae) is a commercially important fisheries resource in Korea. However, no genetic diversity information is available for wild or released hatchery populations. To study the effects of stock enhancement programs and conservation biology, we developed microsatellite DNA markers fromS. inermis. We report the isolation and characterization of seven microsatellite loci, designated KSi221B, KSi239A, KSi25A, KSi252B, KSi193A, KSi259B, and KSi234, isolated using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. To characterize each locus, 30 individuals from a naturalS. inermis population in Yeosu, Korea, were genotyped. All loci except one, KSi193A, were polymorphic with an average of 9.5 alleles per locus (range 2–20). The mean observed and expected heterozygosities were 0.71 (range 0.43–1.00) and 0.76 (range 0.41–0.90), respectively. Significant deviation from Hardy-Weinberg equilibrium was observed at one locus, KSi221B. Such high variability indicates that these microsatellites should provide useful markers for studies of kinship and population genetics.

Molecular, structural, and functional comparison of N lobe and C lobe of the transferrin from rock bream, Oplegnathus fasciatus, with respect to its immune response

Abstract The iron‐withholding strategy of innate immunity is an effective antimicrobial defense mechanism that combats microbial infection by depriving microorganisms of Fe3+, which is important for their growth and propagation. Transferrins (Tfs) are a group of iron‐binding proteins that exert their antimicrobial function through Fe3+ sequestration. The current study describes both structural and functional characteristics of a transferrin ortholog from rock bream Oplegnathus fasciatus (RbTf). The RbTf cDNA possesses an open reading frame (ORF) of 2079 bp encoding 693 amino acids. It has a molecular mass of approximately 74 kDa and an isoelectric point of 5.4. In silico analysis revealed that RbTf has two conserved domains: N‐terminal domain and C‐terminal domain. Pairwise homology analysis and phylogenetic analysis revealed that RbTf shared the highest identity (82.6%) with Dicentrarchus labrax Tf. According to the genomic analysis, RbTf possesses 17 exons and 16 introns, similar to the other orthologs. Here, we cloned the N terminal and C terminal domains of RbTf to evaluate their distinct functional features. Results obtained through the CAS (chrome azurol S) assay confirmed the iron‐binding ability of the RbTf, and it was further determined that the iron‐binding ability of rRbTfN was higher than that of rRbTfC. The antimicrobial functions of the rRbTfN and the rRbTfC were confirmed via the iron‐dependent bacterial growth inhibition assay. Tissue distribution profiling revealed a ubiquitous expression with intense expression in the liver. Temporal assessment revealed that RbTf increased after stimulation of LPS, Edwardsiella tarda, and Streptococcus iniae post injection (p.i.). These findings demonstrated that RbTf is an important antimicrobial protein that can combat bacterial pathogens. HighlightsTransferrin was identified from rock bream (RbTf).N terminal and C terminal domains of RbTf was separately cloned and evaluated their distinct functional features.CAS (chrome azurol S) assay confirmed the iron‐binding ability of the RbTf.Transcriptional level of RbTf was modulated by pathological stress.

Draft Genome Sequence of Beta-Hemolytic Streptococcus iniae KCTC 11634

ABSTRACT Streptococcus iniae is a beta-hemolytic, Gram-positive coccus, which affects a broad range of freshwater and marine fish species, causing substantial economic losses in the aquaculture industry worldwide. Thus, it is very important to derive a complete genome sequence of the bacterium to aid in the development of vaccines and methods for preventing fish streptococcosis and zoonotic infections in humans. Here, we present the draft genome sequence of S. iniae KCTC 11634 (1,955,615 bp, with a G+C content of 36.6%), which contains 1,868 putative coding sequences.

Transcriptome analysis of olive flounder (Paralichthys olivaceus) head kidney infected with moderate and high virulent strains of infectious viral hemorrhagic septicaemia virus (VHSV)

&NA; Olive flounder (Paralichthys olivaceus) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease‐causing VHSV isolates, ADC‐VHS2012‐11 and ADC‐VHS2014‐5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post‐infection using Illumina sequencing. After removing low‐quality sequences, we obtained 144,933,160 high quality reads from thirty‐six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut‐off e‐value of 10−5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV. HighlightsFirst RNA‐seq based transcriptome of moderate and high virulent VHSV‐infected P. olivaceus head kidney.The assembly contig length is more compared to the other olive flounder transcriptome published so far.Genes involved in innate immune response were showed significantly differentially upregulated during day 1 of high virulent and day 3 of moderate virulent.This de novo transcriptome provides useful information for evaluating the molecular mechanisms underlying host‐virus interaction.

Analysis of complete genome and pathogenicity studies of the spring viremia of carp virus isolated from common carp (Cyprinus carpio carpio) and largemouth bass (Micropterus salmoides): An indication of SVC disease threat in Korea

A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea.