Publications by authors named "Ikuo Hirono"

236 Publications

Molecular characterization and expression analysis of Japanese flounder (Paralichthys olivaceus) chemokine receptor CXCR2 in comparison with CXCR1.

Dev Comp Immunol 2021 Feb 26:104047. Epub 2021 Feb 26.

Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan.

Chemokines are categorized into five families; one of the families is the CXC chemokines, which are critical in the pro-inflammatory process. CXC chemokines transmit signals and mediate a cell's biological activities by binding to cell surface receptors known as chemokine receptors (CXCRs). In this study, the CXCR2 from Japanese flounder (Paralichthys olivaceus) (JfCXCR2) was identified and characterized at the molecular level. The JfCXCR2 gene has a 1077 bp open reading frame that encodes a protein of 359 amino acid residues with seven transmembrane domains. Phylogenetic analysis of JfCXCR2 revealed that it belonged to the fish CXCR2 subfamily. Furthermore, JfCXCR2 was compared with the previously identified Japanese flounder CXCR1 (JfCXCR1). The expression analysis of uninfected Japanese flounder showed that JfCXCR1 and JfCXCR2 were expressed in all the tissues and organs tested but mainly in immune-related organs, including the kidney and spleen. Infection by Streptococcus iniae significantly increased the level of JfCXCR1 and JfCXCR2 mRNA in the kidney at days 1 and 3 post-infection. On the other hand, VHSV (viral hemorrhagic septicemia virus) and Edwardsiella tarda infection significantly increased JfCXCR2 mRNA levels in the kidney at days 3 and 6 post-infection, respectively. Conversely, JfCXCR1 expression was not significantly changed by either E. tarda or VHSV infection. Additionally, the peripheral blood leukocytes (PBLs) stimulated by recombinant proteins rCXCL8_L1a and rCXCL8_L1b were found to have significantly increased levels of JfCXCR1 and JfCXCR2 mRNA. Interestingly, even higher levels of JfCXCR1 and JfCXCR2 expression were observed in PBLs stimulated with rCXCL8_L1a and rCXCL8_L1b than in PBLs stimulated with either recombinant protein. These data suggest that bacterial infections may activate JfCXCR1. By contrast, JfCXCR2 may be activated by both bacterial and viral infection to mediate the immune response. These data can contribute to further understanding the functions of CXCR1 and CXCR2 in the fish immune system.
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http://dx.doi.org/10.1016/j.dci.2021.104047DOI Listing
February 2021

Genome Sequence of Vibrio nigripulchritudo Strain TUMSAT-TG-2018, Isolated from Diseased Pacific White Shrimp, .

Microbiol Resour Announc 2020 Dec 3;9(49). Epub 2020 Dec 3.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan

The Gram-negative bacterium is an important shrimp pathogen. Here, we present the genome sequence of TUMSAT-TG-2018, which was isolated from a diseased Pacific white shrimp (). The assembly totaled 6.8 Mbp, consisting of two chromosomes and four plasmids.
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http://dx.doi.org/10.1128/MRA.01206-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714859PMC
December 2020

Cytotoxicity of Streptococcus agalactiae secretory protein on tilapia cultured cells.

J Fish Dis 2020 Oct 10;43(10):1229-1236. Epub 2020 Aug 10.

Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand.

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.
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http://dx.doi.org/10.1111/jfd.13230DOI Listing
October 2020

Characterization of natural antigen-specific antibodies from naïve sturgeon serum.

Dev Comp Immunol 2020 11 4;112:103770. Epub 2020 Jul 4.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo, 108-8477, Japan. Electronic address:

In this study, we isolated and characterized natural antibodies found in serum samples from Bester sturgeon (Huso huso × Acipenser ruthenus). Natural antibodies specifically detected hen egg lysozyme (HEL), keyhole limpet hemocyanin (KLH), and several species of pathogenic bacteria. Interestingly, we detected no antibodies with similar specificity in serum samples from rainbow trout (Oncorhynchus mykiss) or from Japanese flounder (Paralichthys olivaceus). Binding capacity of the sturgeon natural serum antibodies increased slightly at 7 months compared to 3 months after hatching. Antigen-specific antibodies against KLH, Aeromonas hydrophila and Streptococcus iniae were affinity-fractionated from naive sera of Bester sturgeon; specific detection of the corresponding antigens was observed. We conclude that Bester sturgeon are capable of generating unique natural antibodies including those that are pathogen-specific.
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http://dx.doi.org/10.1016/j.dci.2020.103770DOI Listing
November 2020

Draft Genome Sequences of Vibrio atypicus Strains DSM 25292 and TUMSAT1.

Microbiol Resour Announc 2020 Feb 6;9(6). Epub 2020 Feb 6.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan

is a Gram-negative bacterium associated with the digestive tract of penaeid shrimp. Here, we present the draft genome sequences of two strains, DSM 25292 and TUMSAT1. Both assemblies were approximately 4.8 Mbp long, with GC contents of around 44%.
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http://dx.doi.org/10.1128/MRA.01526-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005125PMC
February 2020

Molecular cloning, characterization and gene expression analysis of aminolevulinic acid synthase in Litopenaeus vannamei.

Gene 2020 Apr 1;736:144421. Epub 2020 Feb 1.

Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, Japan. Electronic address:

5-Aminolevulinic acid synthase (ALAS) is the rate-limiting enzyme in the biosynthesis of heme, a prosthetic group that is found in hemoproteins, including those involved in molting. To better understand the roles of ALAS in L. vannamei (LvALAS), we analyzed its sequence and tissue distribution, the effects of age and bacterial infection on its gene expression, and the effects of LvALAS gene silencing. We also examined the expressions of three hemoproteins, the cytochrome oxidase subunit I (COX I) and subunit IV (COX IV) and catalase. Three LvALAS splicing variants were found in the hepatopancreas, with the main splicing variant having an open reading frame that encodes 532 aa. LvALAS transcripts were found in each of the eleven tissues tested in this study, with the highest gene expression in the intestine. The transcript abundances of LvALAS, COX I and COX IV in the hepatopancreas and stomach tended to decrease with age. LvALAS and catalase gene expressions significantly increased in the stomach after V. parahaemolyticus infection. LvALAS gene expression in the hepatopancreas, stomach and intestine (12- and 24-hours post-injection) was relatively lower in dsALAS-injected shrimp than in PBS-injected shrimp. All the PBS-injected shrimp molted after 8-10 days while no molting activity was observed in the dsALAS-injected shrimp group within the 14 days post-injection period. Our results provide evidence that (1) only the housekeeping form of ALAS exists in L. vannamei; LvALAS gene expression (2) decreases with age and (3) increases after bacterial infection; and (4) an ALAS-dependent pathway is necessary for proper molting in L. vannamei.
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http://dx.doi.org/10.1016/j.gene.2020.144421DOI Listing
April 2020

Novel Chimeric Multiepitope Vaccine for Streptococcosis Disease in Nile Tilapia (Oreochromis niloticus Linn.).

Sci Rep 2020 01 17;10(1):603. Epub 2020 Jan 17.

Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngam Wong Wan, Chatuchak, Bangkok, 10900, Thailand.

Streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including Nile tilapia (Oreochromis niloticus Linn.). Vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. Immunoproteomics analysis of S. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. Epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable RNA and protein structure for protein expression. 45F2 and 42E2 were identified as the best candidates for a chimeric multiepitope vaccine. Recombinant plasmids were constructed to produce a recombinant protein vaccine and DNA vaccine system. Overexpressed proteins were determined to be 30 kDa and 25 kDa in the E. coli and TK1 systems, respectively. The efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and DNA vaccine was evaluated in Nile tilapia, followed by S. agalactiae challenge at 1 × 10 CFU/mL. Relative percentage survival (RPS) and cumulative mortality were recorded at approximately 57-76% and 17-30%, respectively. These chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases.
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http://dx.doi.org/10.1038/s41598-019-57283-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969146PMC
January 2020

A Hint of Primitive Mucosal Immunity in Shrimp through Gill C-Type Lectin.

J Immunol 2019 10 13;203(8):2310-2318. Epub 2019 Sep 13.

Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo 108-8477, Japan; and

Lectins are found in most living organisms, providing immune surveillance by binding to carbohydrate ligands. In fishes, C-type lectins were isolated from mucus of respiratory organs (skin and gills), where they aid the mucosal immune response in regulating microbiota and suppressing pathogens. In shrimp, however, no mucosal immunity or any form of gill-specific immune defense has been reported, and most identified C-type lectins are associated with hemocyte cellular and humoral responses. Interestingly, our microarray analysis revealed the localization of highly expressed novel biodefense genes in gills, among which is gill C-type lectin (MjGCTL), which we previously reported. Gill mucus collected from displayed similar bacterial agglutination ability as observed with recombinant MjGCTL. This agglutinating ability can be attributed to endogenous MjGCTL (nMjGCTL) detected in gill mucus, which was confirmed with an agglutination assay using purified nMjGCTL from gills. In addition, nMjGCTL also promoted in vivo bacterial phagocytosis by hemocytes. In vivo knockdown of MjGCTL resulted in a compromised immune system, which was manifested by impaired agglutination capacity of gill mucus and downregulation of the gill antimicrobial peptides, crustin and penaeidin. Shrimp immunocompromised by MjCGTL knockdown, apparently lost the ability to respond to attaching and penetrating bacteria. This was evident as increased total bacteria and counts in both gills and hemolymph, which were correlated with low survival during a bacterial challenge. These results reveal immune defense by shrimp gills resembling a primitive form of mucosal immunity.
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http://dx.doi.org/10.4049/jimmunol.1900156DOI Listing
October 2019

Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease.

J Fish Dis 2019 Aug 21;42(8):1125-1132. Epub 2019 May 21.

Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, Japan.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.
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http://dx.doi.org/10.1111/jfd.13024DOI Listing
August 2019

ICTV Virus Taxonomy Profile: Nimaviridae.

J Gen Virol 2019 07 29;100(7):1053-1054. Epub 2019 Mar 29.

6​Pathology and Molecular Systematics Team, Centre for Environment, Fisheries andAquaculture Science (Cefas), Weymouth, DT4 8UB, UK.

The family Nimaviridae includes the single species White spot syndrome virus, isolates of which infect a wide range of aquatic crustaceans and cause substantial economic losses. Virions are ellipsoid to bacilliform with a terminal thread-like extension. The circular dsDNA genome is 280-307 kbp with several homologous repeat regions. More than 80 structural and functional proteins have been characterized from 531 ORFs. White spot syndrome is a highly lethal, contagious disease associated with white spot syndrome virus infection of shrimps. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Nimaviridae, which is available at www.ictv.global/report/nimaviridae.
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http://dx.doi.org/10.1099/jgv.0.001248DOI Listing
July 2019

Identification and expression analysis of Fc receptor-like proteins in Japanese flounder (Paralichthys olivaceus).

Fish Shellfish Immunol 2019 Apr 4;87:82-86. Epub 2019 Jan 4.

Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan. Electronic address:

Fc receptors (FcRs) are specific to the Fc portion of immunoglobulin (Ig) molecules. Here, four Fc receptor-like proteins, JF-FcR-like protein 1-4, were identified in Japanese flounder. Their open reading frames encoded 358, 255, 519 and 441 amino acid residues, respectively. JF-FcR-like protein mRNAs were mainly detected in kidney and spleen of healthy fish. Injection of formalin-killed cells (FKCs) of Edwardsiella tarda significantly increased the spleen mRNA levels of JF-FcR-like protein 1 but not the other JF-FcR-like proteins. Injection of FKC of Streptococcus iniae did not significantly affect any of the JF-FcR-like protein mRNAs. These findings suggest that the FcR-like proteins have different involvements in pathogen responses.
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http://dx.doi.org/10.1016/j.fsi.2019.01.002DOI Listing
April 2019

Hematopoietic tissue of Macrobrachium rosenbergii plays dual roles as a source of hemocyte hematopoiesis and as a defensive mechanism against Macrobrachium rosenbergii nodavirus infection.

Fish Shellfish Immunol 2019 Mar 13;86:756-763. Epub 2018 Dec 13.

Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand; Nakhonsawan Campus, Mahidol University, Nakhonsawan, 60130, Thailand. Electronic address:

White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar), are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii.
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http://dx.doi.org/10.1016/j.fsi.2018.12.021DOI Listing
March 2019

Comparative genomics inferred two distinct populations of piscine pathogenic Streptococcus agalactiae, serotype Ia ST7 and serotype III ST283, in Thailand and Vietnam.

Genomics 2019 12 17;111(6):1657-1667. Epub 2018 Nov 17.

Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand.

The genomes of Streptococcus agalactiae (group B streptococcus; GBS) collected from diseased fish in Thailand and Vietnam over a nine-year period (2008-2016) were sequenced and compared (n = 21). Based on capsular serotype and multilocus sequence typing (MLST), GBS isolates are divided into 2 groups comprised of i) serotype Ia; sequence type (ST)7 and ii) serotype III; ST283. Population structure inferred by core genome (cg)MLST and Bayesian clustering analysis also strongly indicated distribution of two GBS populations in both Thailand and Vietnam. Deep phylogenetic analysis implied by CRISPR array's spacer diversity was able to cluster GBS isolates according to their temporal and geographic origins, though ST7 has varying CRISPR1-spacer profiles when compared to ST283 strains. Based on overall genotypic features, Thai ST283 strains were closely related to the Singaporean ST283 strain causing foodborne illness in humans in 2015, thus, signifying zoonotic potential of this GBS population in the country.
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http://dx.doi.org/10.1016/j.ygeno.2018.11.016DOI Listing
December 2019

Crustacean Genome Exploration Reveals the Evolutionary Origin of White Spot Syndrome Virus.

J Virol 2019 02 17;93(3). Epub 2019 Jan 17.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan

White spot syndrome virus (WSSV) is a crustacean-infecting, double-stranded DNA virus and is the most serious viral pathogen in the global shrimp industry. WSSV is the sole recognized member of the family , and the lack of genomic data on other nimaviruses has obscured the evolutionary history of WSSV. Here, we investigated the evolutionary history of WSSV by characterizing WSSV relatives hidden in host genomic data. We surveyed 14 host crustacean genomes and identified five novel nimaviral genomes. Comparative genomic analysis of identified 28 "core genes" that are ubiquitously conserved in ; unexpected conservation of 13 uncharacterized proteins highlighted yet-unknown essential functions underlying the nimavirus replication cycle. The ancestral gene set contained five baculoviral infectivity factor homologs and a sulfhydryl oxidase homolog, suggesting a shared phylogenetic origin of and insect-associated double-stranded DNA viruses. Moreover, we show that novel gene acquisition and subsequent amplification reinforced the unique accessory gene repertoire of WSSV. Expansion of unique envelope protein and nonstructural virulence-associated genes may have been the key genomic event that made WSSV such a deadly pathogen. WSSV is the deadliest viral pathogen threatening global shrimp aquaculture. The evolutionary history of WSSV has remained a mystery, because few WSSV relatives, or nimaviruses, had been reported. Our aim was to trace the history of WSSV using the genomes of novel nimaviruses hidden in host genome data. We demonstrate that WSSV emerged from a diverse family of crustacean-infecting large DNA viruses. By comparing the genomes of WSSV and its relatives, we show that WSSV possesses an expanded set of unique host-virus interaction-related genes. This extensive gene gain may have been the key genomic event that made WSSV such a deadly pathogen. Moreover, conservation of insect-infecting virus protein homologs suggests a common phylogenetic origin of crustacean-infecting and other insect-infecting DNA viruses. Our work redefines the previously poorly characterized crustacean virus family and reveals the ancient genomic events that preordained the emergence of a devastating shrimp pathogen.
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http://dx.doi.org/10.1128/JVI.01144-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340018PMC
February 2019

Identification of an anti-lipopolysaccharide factor AV-R isoform (LvALF AV-R) related to Vp_PirAB-like toxin resistance in Litopenaeus vannamei.

Fish Shellfish Immunol 2019 Jan 4;84:178-188. Epub 2018 Oct 4.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan. Electronic address:

Acute hepatopancreatic necrosis disease (AHPND) is a shrimp farming disease, caused by the pathogenic Vibrio parahaemolyticus carrying a plasmid encoding Vp_PirAB-like toxins. Formalin-killed cells of V. parahaemolyticus AHPND-causing strain D6 (FKC-VpD6) were used to select Vp_PirAB-like toxin-resistant Litopenaeus vannamei by oral administration. Stomach and hepatopancreas tissues of shrimps that survived for one week were subjected to RNA sequencing. Differentially expressed genes (DEGs) between surviving shrimp, AHPND-infected shrimp, and normal shrimp were identified. The expressions of 10 DEGs were validated by qPCR. Only one gene (a gene homologous to L. vannamei anti-lipopolysaccharide factor AV-R isoform (LvALF AV-R)) was expressed significantly more strongly in the hepatopancreas of surviving shrimp than in the other groups. Significantly higher expression of LvALF AV-R was also observed in shrimp that survived two other trials of FKC-VpD6 selection. Recombinant ALF AV-R bound to LPS, PGN, Gram-negative bacteria, and some Gram-positive bacteria in ELISAs. ALF AV-R recombinant protein did not interact with native Vp_PirAB-like toxin in an ELISA or a Far-Western blot. For L. vannamei orally fed ALF AV-R protein for 3 days, the survival rate following challenge with VpD6-immersion was not significantly different from that of shrimp fed two control diets. These results suggest that LvALF AV-R expression was induced in the hepatopancreas of shrimp in response to the presence of Vp_PirAB-like toxin, although other factors might also be involved in the resistance mechanism.
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http://dx.doi.org/10.1016/j.fsi.2018.10.005DOI Listing
January 2019

Adjuvant effects on protection and immune response of Japanese flounder immunized by the formalin-killed cells of Edwardsiella tarda.

Fish Shellfish Immunol 2019 Jan 27;84:120-123. Epub 2018 Sep 27.

Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan.

We evaluated the effects of Freund's adjuvants (FCA/FIA) on protection and immune response of Japanese flounder Paralichthys olivaceus immunized by the formalin-killed cell (FKC) of Edwardsiella tarda. Combination of FKC and FCA/FIA did not confer protection against the challenge, while they significantly induced higher antibody titers than that of FKC only. The suppression of FKC-dependent induction of interferon γ (IFNγ) mRNA levels by FCA/FIA was observed by gene expression profiling. Similarly, interleukin (IL)-12 p35 mRNA levels were not detected after FKC+FCA or +FIA. These results suggest that the mineral oil in Freund's adjuvants might suppress the signaling pathway(s) that induce IFNγ and IL-12 gene expression.
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http://dx.doi.org/10.1016/j.fsi.2018.09.071DOI Listing
January 2019

Effects of 5-Aminolevulinic Acid on Gene Expression, Immunity, and ATP Levels in Pacific White Shrimp, Litopenaeus vannamei.

Mar Biotechnol (NY) 2018 Dec 25;20(6):829-843. Epub 2018 Aug 25.

Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, Japan.

With the emergence of several infectious diseases in shrimp aquaculture, there is a growing interest in the use of feed additives to enhance shrimp immunity. Recently, the use of 5-aminolevulinic acid (5-ALA), a non-protein amino acid that plays a rate-limiting role in heme biosynthesis, has received attention for its positive effect on immunity in livestock animals. To evaluate the effect of 5-ALA in the Pacific white shrimp, Litopenaeus vannamei, we conducted microarray analysis, a Vibrio parahaemolyticus immersion challenge test, an ATP level assay, and gene expression analysis of some hemoproteins and genes associated with heme synthesis and degradation. Out of 15,745 L. vannamei putative genes on the microarray, 101 genes were differentially expressed by more than fourfold (p < 0.05) between 5-ALA-supplemented and control shrimp hepatopancreas. 5-ALA upregulated 99 of the 101 genes, 41 of which were immune- and defense-related genes based on sequence homology. Compared to the control, the 5-ALA-supplemented group had a higher survival rate in the challenge test, higher transcript levels of porphobilinogen synthase, ferrochelatase, catalase, nuclear receptor E75, and heme oxygenase-1 and higher levels of ATP. These findings suggest that dietary 5-ALA enhanced the immune response of L. vannamei to V. parahaemolyticus, upregulated immune- and defense-related genes, and enhanced aerobic energy metabolism, respectively. Further studies are needed to elucidate the extent of 5-ALA use in shrimp culture.
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http://dx.doi.org/10.1007/s10126-018-9852-2DOI Listing
December 2018

Comparative sequence analysis of crustin isoform MjCRS7 and MjWFDC-like gene from kuruma shrimp Marsupenaeus japonicus shows variant of the WFDC domain.

Infect Genet Evol 2018 10 7;64:139-148. Epub 2018 Jun 7.

Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan. Electronic address:

Crustins are well known cysteine-rich cationic antimicrobial peptides (AMPs) in crustaceans that have WFDC [WAP (whey acidic protein) four-disulfide core] domain at the carboxyl terminus. Proteins containing a WFDC domain have been discovered in many invertebrates and vertebrates. Although, there have been many WFDC domain containing nucleotide sequences found in NCBI GenBank database, their distinct sequential characteristics and their role in the innate immune system is not well understood. Here, we identified a new crustin isoform from Marsupenaeus japonicus by transcriptome analysis. The full-length cDNA of this isoform (MjCRS7) consists of 537 bp that include a 489 bp open reading frame (ORF) encoding 162 deduced amino acids (aa). The sequence contains the eight conserved cysteine residues characteristic of the WFDC domain. A phylogenetic analysis showed that MjCRS7 is a type II crustin. We also identified the full-length cDNA of a M. japonicus MjWFDC-like gene. MjWFDC-like has a 543 bp ORF encoding 180 aa. In an RT-PCR analysis, MjCRS7 and MjWFDC-like transcripts were mainly detected in gill tissue. An alignment of MjCRS7 and MjWFDC-like with previously reported M. japonicus crustin isoform 1-5 (MjCRS1-5) showed variation in the WFDC-like domain. Neither of the genes was responsive to Vibrio parahaemolyticus, Vibrio penaeicida or white spot syndrome virus (WSSV) either by immersion or injection challenge test. Although crustins are mainly antimicrobial peptides, the present results suggest that MjCRS7 may have other roles in M. japonicus.
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http://dx.doi.org/10.1016/j.meegid.2018.06.008DOI Listing
October 2018

Rapid diagnosis of three shrimp RNA viruses using RT-PCR-DNA chromatography.

J Fish Dis 2018 May 28. Epub 2018 May 28.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan.

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http://dx.doi.org/10.1111/jfd.12821DOI Listing
May 2018

A novel white spot syndrome virus protein WSSV164 controls prophenoloxidases, PmproPOs in shrimp melanization cascade.

Dev Comp Immunol 2018 09 4;86:109-117. Epub 2018 May 4.

Aquatic Molecular Genetics and Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand. Electronic address:

Melanization, mediated by the prophenoloxidase (proPO)-activating system, is an important innate immune response in invertebrates. The implication of the proPO system in antiviral response and the suppression of host proPO activation by the viral protein have previously been demonstrated in shrimp. However, the molecular mechanism of viral-host interactions in the proPO cascade remains largely unexplored. Here, we characterized the viral protein, namely, WSSV164, which was initially identified from the forward suppression subtractive hybridization (SSH) cDNA library of the PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon that was challenged with white spot syndrome virus (WSSV). Using the yeast two-hybrid system, WSSV164 was found to interact with the PmproPO2 protein. The subsequent validation assay by co-immunoprecipitation revealed that WSSV164 directly bound to both PmproPO1 and PmproPO2. The gene silencing experiment was carried out to explore the role of WSSV164 in the control of the proPO pathway in shrimp, and the results showed that suppression of WSSV164 can restore PO activity in WSSV-infected shrimp hemolymph. The recombinant proteins of PmproPO1 and PmproPO2 were produced in Sf-9 cells and were shown to be successfully activated by exogenous trypsin and endogenous serine proteinases from shrimp hemocyte lysate supernatant (HLS), yielding PO activity in vitro. Moreover, the activated PO activity in shrimp HLS was dose-dependently reduced by the recombinant WSSV164 protein, suggesting that WSSV164 may interfere with the activation of the proPO system in shrimp. Taken together, these results suggest an alternative infection route of WSSV through the encoded viral protein WSSV164 that binds to the PmproPO1 and PmproPO2 proteins, interfering with the activation of the melanization cascade in shrimp.
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http://dx.doi.org/10.1016/j.dci.2018.05.005DOI Listing
September 2018

White spot syndrome virus (WSSV) suppresses penaeidin expression in Marsupenaeus japonicus hemocytes.

Fish Shellfish Immunol 2018 Jul 22;78:233-237. Epub 2018 Apr 22.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan. Electronic address:

Penaeidins are a unique family of antimicrobial peptides specific to penaeid shrimp and have been reported mainly function as anti-bacterial and anti-fungal. In order to investigate whether penaeidins could also respond to virus or not, we examined the effect of WSSV on MjPen-II (penaeidin in kuruma shrimp, Marsupenaeus japonicus) expression. In the control group, MjPen-II transcript level can be detected in almost all test tissues but was expressed most strongly in hemocytes. After WSSV infection, MjPen-II transcript level was significantly downregulated in hemocytes. Moreover, the proportion of MjPen-II+ hemocytes was not significantly different between non-infected and WSSV-infected shrimp, but the number of MjPen-II+ highly expressing hemocytes decreased after infection. In addition, MjPen-II was observed in the cytoplasm of granule-containing hemocytes. These results suggest that WSSV suppresses MjPen-II expression in hemocytes.
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http://dx.doi.org/10.1016/j.fsi.2018.04.045DOI Listing
July 2018

The immune functions of sessile hemocytes in three organs of kuruma shrimp Marsupenaeus japonicus differ from those of circulating hemocytes.

Fish Shellfish Immunol 2018 Jul 21;78:109-113. Epub 2018 Apr 21.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan. Electronic address:

Shrimp, as invertebrates, have an open vasculature that allows circulating hemocytes to infiltrate the tissues, where they are referred to as sessile hemocytes. Sessile hemocytes are known to express immune-related genes, but it is not known whether their functions differ from those of circulating hemocytes. To answer this question, we enriched them from suspensions of different tissues using discontinuous density gradient centrifugation and analyzed their transcripts by RNA-seq. The results suggest that circulating hemocytes and sessile hemocytes of the gills are in a state that could react quickly to pathogens, immune-related genes expression of sessile hemocytes differ from circulating hemocytes, and the gills, heart and lymphoid organs have cells that express immune-related genes that are different from hemocytes.
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http://dx.doi.org/10.1016/j.fsi.2018.04.036DOI Listing
July 2018

The complete mitochondrial genome sequence of the sakura shrimp, (Crustacea, Decapoda, ).

Mitochondrial DNA B Resour 2018 Apr 2;3(1):444-445. Epub 2018 Apr 2.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan.

Here, we present the complete mitochondrial genome of the sakura shrimp, (Crustacea, Decapoda, ). The circular genome is 16,087 base pairs in length and contains 13 protein-coding genes, 22 tRNA genes, and two rRNA genes. The nucleotide composition of the mitogenome is biased towards A + T (69.9%). The gene arrangement was identical to penaeid shrimps. Maximum likelihood phylogenetic analysis using the nucleotide sequences of 13 protein-coding genes placed next to , supporting the conventional taxonomic relationship of and .
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http://dx.doi.org/10.1080/23802359.2018.1450671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7800779PMC
April 2018

Gills specific type 2 crustin isoforms: Its molecular cloning and characterization from kuruma shrimp Marsupenaeus japonicus.

Dev Comp Immunol 2018 08 27;85:25-30. Epub 2018 Mar 27.

Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan. Electronic address:

Crustins are diverse group of antimicrobial peptides (AMPs) that have numerous isoforms mainly identified from hemocytes in decapods crustacean. However, little is known about its presence solely in gills tissue. In this study, we found two new crustin isoforms MjCRS8 and MjCRS9 by using transcriptome analysis from gills. Open reading frame of MjCRS8 and MjCRS9 were 593 bp and 459 bp encoding 197aa and 152aa, respectively. Tissue distribution analysis indicated that both MjCRS8 and MjCRS9 are expressed only in gills tissue. Multiple sequence alignment and phylogenetic analysis with previously reported crustin suggested that both MjCRS8 and MjCRS9 belong to type 2 crustin family. Experimental infection was conducted against Vibrio parahaemolyticus and white spot syndrome virus (WSSV) by immersion test. However, no significant upregulation was observed.
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http://dx.doi.org/10.1016/j.dci.2018.03.018DOI Listing
August 2018

Class B CpG-ODN2006 is highly associated with IgM and antimicrobial peptide gene expression through TLR9 pathway in yellowtail Seriola lalandi.

Fish Shellfish Immunol 2018 Jun 19;77:71-82. Epub 2018 Mar 19.

Grupo de Inmunología y Vacunología, Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Instituto Politécnico Nacional 195, Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico. Electronic address:

The purpose of this study was to characterize the TLR9 gene from yellowtail (Seriola lalandi) and evaluate its functional activity using the class B Cytosine-phosphate-guanine-oligodeoxynucleotide2006 (CpG-ODN2006) in an in vivo experiment after one-week immunostimulation. The gene expressions of TLR9, Immunoglobulin M (IgM), antimicrobial peptides and cytokines were evaluated by real time PCR, and humoral immune parameters were analyzed in serum. The TLR9 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. The yellowtail full-length cDNA sequence of SlTLR9 was 3789 bp in length, including a 66-bp 5'-untranslated region (UTR), a 3'-UTR of 528 bp, and an open reading frame (ORF) of 3192 bp translatable to 1064 amino acid showing a high degree of similarity with the counterparts of other fish species and sharing common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR9 mRNA in all analyzed tissues; the highest levels were observed in intestine, liver and spleen where they play an important role in the fish immune system. The expression levels of TLR9 after B class CpG-ODN2006 (the main TLR9-agonist) was significantly up-regulated in all analyzed tissues, with the high expression observed in spleen followed by intestine and skin. The CpG-B has been shown as a potent B cell mitogen, and interestingly, IgM mRNA transcript was up-regulated in spleen and intestine, which was highly correlated with TLR9 after CpG-ODN2006 stimulation. The antimicrobial peptides, piscidin and NK-lysine, were up-regulated in spleen and gill after CpG-ODN2006 injection with a high correlation (r ≥ 0.82) with TLR9 gene expression. Cytokine genes were up-regulated in spleen, intestine and skin after CpG-ODN was compared with the control group. No significant correlation was observed between TLR9 and IL-1β, TNF-α and Mx gene expressions. The results showed that CpG-ODN2006 intraperitoneal injection enhanced lysozyme, peroxidase and superoxide dismutase activities in serum and demonstrated that CpG-ODN2006 can induce a specific immune response via TLR9 in which IgM and antimicrobial peptides must have an important role in the defense mechanisms against infections in yellowtail.
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http://dx.doi.org/10.1016/j.fsi.2018.03.025DOI Listing
June 2018

A novel white spot syndrome virus-induced gene (MjVIG1) from Marsupenaeus japonicus hemocytes.

Fish Shellfish Immunol 2018 Jun 19;77:46-52. Epub 2018 Mar 19.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo, 108-8477, Japan. Electronic address:

cDNA of a newly recognized white spot syndrome virus (WSSV)-induced gene (MjVIG1) was characterized from Marsupenaeus japonicus hemocytes; this gene encodes a protein that lack similarity to any known characterized protein. To identify this novel gene, we mainly conducted transcript level analysis, immunostaining and flow cytometry after WSSV infection. MjV1G1 transcript levels were also measured after Yellow head virus (YHV) and Vibrio parahaemolyticus infection tests. In non-infected and WSSV-infected shrimp, MjVIG1 was observed in granule-containing hemocytes. In addition, the MjVIG1 transcript level and ratio of MjVIG1-positive hemocytes both significantly increased, and number of MjVIG1-positive hemocytes slightly increased after WSSV infection. In contrast, MjVIG1 transcript level did not change after YHV and V. parahaemolyticus infection. These results indicated that MjVIG1 might be a WSSV-specific induced gene in M. japonicus hemocytes.
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http://dx.doi.org/10.1016/j.fsi.2018.03.026DOI Listing
June 2018

Draft Genome Sequence of Vibrio penaeicida Strain TUMSAT-NU1, Isolated from Diseased Shrimp in Japan.

Genome Announc 2018 Mar 15;6(11). Epub 2018 Mar 15.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan

is a bacterial pathogen of cultured shrimp. The draft genome sequence of strain TUMSAT-NU1 consists of 100 scaffolds with a total of 6.41 Mbp. We identified possible virulence factors, and we found that and are closely related.
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http://dx.doi.org/10.1128/genomeA.00153-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854779PMC
March 2018

RNA-seq identifies integrin alpha of kuruma shrimp Marsupenaeus japonicus as a candidate molecular marker for phagocytic hemocytes.

Dev Comp Immunol 2018 04 16;81:271-278. Epub 2017 Dec 16.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo, 108-8477, Japan. Electronic address:

Phagocytosis is main cellular immunity, however, it is still unknown or debated upon which types of hemocyte contributes phagocytosis in penaeid shrimps. The hemocyte characterization in kuruma shrimp have been mainly performed based on its morphology by microscopic observation. Therefore, establishment of molecular markers to distinguish phagocytic hemocytes is required. In this study, using magnetic fluorescent beads, we enriched phagocytic hemocytes and conducted RNA-seq analysis between total and enriched phagocytic hemocytes. The data demonstrated functional difference between total and phagocytic hemocytes. In addition, a transcript homologous to integrin-alpha was highly expressed in phagocytic hemocytes, and named Mj-Intgα. Using anti-serum against Mj-Intgα revealed that around 60% of total hemocytes and more than 90% of phagocytic hemocytes showed positive for Mj-Intgα. This study presents Mj-Intgα as a candidate molecular marker for future functional characterization of hemocytes.
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http://dx.doi.org/10.1016/j.dci.2017.12.014DOI Listing
April 2018

A novel viral responsive protein (MjVRP) from Marsupenaeus japonicus haemocytes is involved in white spot syndrome virus infection.

Fish Shellfish Immunol 2017 Nov 18;70:638-647. Epub 2017 Sep 18.

Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan. Electronic address:

A viral responsive protein (MjVRP) was characterized from Marsupenaeus japonicus haemocytes. In amino acid homology and phylogenetic tree analyses, MjVRP clustered in the same group with the viral responsive protein of Penaeus monodon (PmVRP15), showing 34% identity. MjVRP transcripts were mainly expressed in haemocytes and the lymphoid organ. Western blotting likewise showed that MjVRP was strongly expressed in haemocytes and the lymphoid organ. Immunostaining detected MjVRP within the cytosol next to the perinuclear region in some haemocytes. Experimental challenge with white spot syndrome virus (WSSV) significantly up-regulated the mRNA level of MjVRP in the M. japonicus haemocytes at 6 and 48 h. Flow cytometry and indirect immunofluorescence assays revealed that the ratio of MjVRP haemocytes significantly increased 24 and 48 h post-WSSV infection. These results suggest that MjVRP haemocytes have a supporting role in the pathogenesis of WSSV.
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http://dx.doi.org/10.1016/j.fsi.2017.09.045DOI Listing
November 2017

Complete Genome Sequence of the Lytic Giant Bacteriophage pT24 Infecting spp., Isolated from a Shrimp Culture Pond.

Genome Announc 2017 Jul 6;5(27). Epub 2017 Jul 6.

School of Marine Science, Tokyo University of Marine Science and Technology, Tokyo, Japan

The lytic bacteriophage pT24, which infects spp., was isolated from the water of a whiteleg shrimp () culture pond in Thailand. This giant bacteriophage with myovirus morphology comprised 234,670 bp with 296 predicted genes.
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http://dx.doi.org/10.1128/genomeA.00081-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502841PMC
July 2017