Reiko Nozaki

Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus

The cysteine‐rich peptide hepcidin is known to be an antimicrobial peptide and iron transport regulator that has been found in both fish and mammals. Recently, we found two different types (designated Hep‐JF1 and Hep‐JF2) of hepcidin cDNA in the Japanese flounder, Paralichthys olivaceus, by expressed sequence tag analysis. The identity of amino acid sequences between Hep‐JF1 and Hep‐JF2 was 51%. The Hep‐JF1 and Hep‐JF2 genes both consist of three exons and two introns, and both exist as single copies in the genome. The predicted mature regions of Hep‐JF1 and Hep‐JF2 have six and eight Cys residues, respectively. The first Cys residue of Hep‐JF1 was deleted and the second was replaced with Gly. The number and positions of Cys residues in Hep‐JF2 are the same as they are in human Hep. Hep‐JF1 is specifically expressed in liver while the expression of Hep‐JF2 was detected from gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach. Gene expression of Hep‐JF1 in liver decreased during experimental iron (iron‐dextran) overload. Expression of Hep‐JF1 in liver was decreased by injecting fish with iron‐dextran and increased by injecting lipopolysaccharide. Iron overload did not significantly affect expression of Hep‐JF2 in liver but it did increase expression of Hep‐JF2 in kidney. Lipopolysaccharide injection increased expression of Hep‐JF2 in both liver and kidney. In liver, some cells expressed both Hep‐JF1 and Hep‐JF2 while some other cells expressed just one of them. Synthesized Hep‐JF2 peptide showed antimicrobial activity, while synthesized Hep‐JF1 peptide did not against several bacteria including fish‐pathogenic bacteria used in this study.

Draft Genome Sequences of Six Strains of Vibrio parahaemolyticus Isolated from Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Disease Shrimp in Thailand

ABSTRACT Some strains of Vibrio parahaemolyticus cause acute hepatopancreatic necrosis disease (AHPND) in shrimp. We sequenced 3 AHPND and 3 non-AHPND strains and found that all of them lacked the pathogenicity island relevant to human infection. A unique sequence encoding a type IV pilus/type IV secretion system was found in 3 AHPND strains.

EST analysis on adipose tissue of rainbow trout Oncorhynchus mykiss and tissue distribution of adiponectin

Although energy metabolism in mammals is critically regulated by adipokines from adipocytes, it is unclear whether this is the case in fish as well. In this study, over 30,000 expressed sequence tags (ESTs) were obtained from adipose tissue in rainbow trout Oncorhynchus mykiss peritoneal cavity and searched for genes possibly related to lipid metabolism. Large numbers of ESTs encoded digestive enzymes and hormones usually found in the pancreas in higher vertebrates, consistent with the fact that pancreatic cells are dispersed in the adipose tissue. Many ESTs encoded apolipoprotein C-I, fatty acid-binding proteins and lymphocyte G0/G1 switch protein 2, which function in lipid transport, fatty acid accumulation and adipocyte differentiation, respectively. None of the ESTs encoded adipokines. We therefore obtained a cDNA encoding adiponectin, an adipokine that regulates oxidation of glucose and lipids in peripheral tissues, using rainbow trout ESTs in the public database. Real-time RT-PCR analyses revealed that its transcript levels were high in muscle and quite low in adipose tissue. These results strongly suggest that adipocytes of rainbow trout and possibly other fish species, unlike those of mammals, are not involved in the production of adipokines.

Identification of novel interleukin 1 beta family genes in Japanese flounder Paralichthys olivaceus

Gene in the interleukin 1 family plays a central role in the regulation of immune and inflammatory responses. Here, we describe two novel IL-1-like gene in Japanese flounder Paralichthys olivaceus (jfIL1-L1 and jfIL1-L2). jfIL1β-L1 was homologous to Nile Tilapia IL-1β-like gene and Arctic char IL-1, and jfIL1β-L2 showed homology to hypothetical protein LOC100699119 of Nile Tilapia and rainbow trout Oncorhynchus mykiss IL-1 receptor agonist (RA). The deduced amino acids sequences of these IL-1β-like genes showed very low identities to the Japanese flounder IL-1 (jfIL-1). Phylogenetic analysis confirmed that jfIL1β-L1 and -L2 were distinct from jfIL-1. The gene encoding the predicted ORF of jfIL1β-L1 and -L2 is divided into 6 exons and 7 exons, respectively. Transcripts of jfIL1β-L1 were detected in gills, intestine, kidney and spleen, and those of jfIL1β-L2 were detected in gills, intestine and spleen. The mRNA levels of jfIL1β-L1 and -L2 were not effect or slightly decreased by treatment with LPS and the formalin-killed cells of Edwardsiella tarda whilst mRNA levels of jfIL1β were significantly increased in the kidney and spleen at 6 h by these treatments.

Cloning and expression analysis of three novel CC chemokine genes from Japanese flounder (Paralichthys olivaceus).

Chemokines are small cytokines secreted by various cell types. They not only function in cell activation, differentiation and trafficking, but they also have influences on many biological processes. In this study, three novel CC chemokine genes Paol-SCYA105, 106 and 107 in Japanese flounder (Paralichthys olivaceus) were cloned and characterized. Paol-SCYA105 was mainly detected in gill, kidney and spleen, Paol-SCYA106 was detected in all tissues examined and Paol-SCYA107 was mainly detected in the spleen and kidney. Paol-SCYA105 and Paol-SCYA106 gene expressions peaked in kidney at day 3 after viral hemorrhagic septicemia virus infection and decreased at day 6, but Paol-SCYA106 still remained at a high level at day 6. Paol-SCYA107 gene expression was significantly up-regulated in kidney at day 6 after viral hemorrhagic septicemia virus infection. In response to infection by Gram-negative Edwardsiella tarda and Gram-positive Streptococcus iniae in kidney, only Paol-SCYA106 gene expression significantly increased. Together, these results indicate that these three novel CC chemokines are involved in the immune response against pathogen infections.

Virulence of Acute Hepatopancreatic Necrosis Disease PirAB-like Relies on Secreted Proteins Not on Gene Copy Number.

To investigate the virulence of the Vp_PirAB‐like genes in Vibrio parahaemolyticus‐ acute hepatopancreatic necrosis disease (AHPND)‐causing strain and the factors that are associated with the virulence level.

Two hemocyte sub-populations of kuruma shrimp Marsupenaeus japonicus

&NA; Hemocytes in the circulating hemolymph play important roles for immune responses in shrimp. Previous studies on immune responses by hemocytes in penaeid shrimp were based on gene expression analyses of the entire population of hemocytes and thus may have missed different immune responses of different hemocyte sub‐populations. In this study, we separated hemocytes into two sub‐populations by Percoll gradient centrifugation, morphological characteristics of each population were then analyzed by May–Giemsa staining, flow cytometry, and FACSCalibur. Results showed hemocytes were divided into an upper layer basophilic, and lower layer of eosinophilic hemocytes. Basophilic hemocytes were larger in size compared to eosinophilic hemocytes, which were more granulated than the basophilic hemocytes. Transcriptome analysis was then conducted through RNA‐seq analysis by Miseq, which revealed 16 differentially‐expressed transcripts between the two sub‐populations. In the upper‐layer, the highly expressed transcripts that were homologous to immune‐related genes that suggest hemocytes from this layer may play as the regulator of immune system and control the action of other cells to eliminate pathogen. On the other hand, transcripts that were highly expressed in the lower‐layer were homologous to the antimicrobial peptide (AMP) crustin, which supports that hemocytes on this layer have granules as crustins are normally secreted from hemocyte granules. The high expression of crustin in the lower‐layer also provides insight on the mechanism of the anti‐microbial function, where hemocytes produce and store AMPs in its granules. These differentially expressed genes are potential hemocyte molecular markers, and among them we identified one of the highly expressed genes in the hemocytes from the upper‐layer (c11736_g1) to be a promising candidate molecular marker predicted to be a surface molecule, which is a common characteristic for molecular markers. HighlightsMorphological and molecular characterization of two hemocyte sub‐populations.Differentially expressed transcripts between the two sub‐populations were identified.c11736_g1 possess structural characteristics of a typical molecular marker.

Detection of acute hepatopancreatic necrosis disease strain of Vibrio parahaemolyticus using loop-mediated isothermal amplification

Acute hepatopancreatic necrosis disease (AHPND) is caused by specific strains of Vibrio parahaemolyticus that have a virulent plasmid carrying toxin genes (Gomez-Gil et al. 2014; Kondo et al. 2014; Yang et al. 2014; Han et al. 2015). Presently, AHPND can be detected by conventional PCR (http:// www.enaca.org/). An improved PCR method using a primer set that targets JHE-like toxin PirA-like of V. parahaemolyticus (TUMSAT-Vp3) was recently reported (Tinwongger et al. 2014). However, PCR takes several hours and requires an expensive thermal cycler. Alternatively, DNA can be amplified under isothermal conditions in only 1 h by a new method called loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000; Mori et al. 2001). LAMP generates a large amount of a series of stem-loop amplicons with various lengths from a small amount of template (Notomi et al. 2000; Mori et al. 2001). LAMP requires four primers, which give it a high specificity for detection. LAMP may not be useful for other PCR applications such as cloning, but it is well suited for disease diagnosis (Savan et al. 2005). LAMP has been used to detect shrimp diseases caused by viruses such as white spot syndrome virus (Kono et al. 2004), yellow head virus (Mekata et al. 2006), infectious hypodermal and hematopoietic necrosis virus (Sun et al. 2006), Taura syndrome virus (Kiatpathomchai et al. 2008) and myonecrosis virus (Puthawibool et al. 2009). We used six V. parahaemolyticus strains isolated from shrimp farms in Thailand (Tinwongger et al. 2014): two AHPND (E2 and D6) strains and four non-AHPND (N7, N10, FP11 and FP14) strains. Total genomic DNA was extracted by the CTAB method (Sambrook & Russel 2001). A set of primers specific to AHPND strain was designed based on the JHE-like toxin PirA-like and PirB-like (toxin PirAB-like) of the plasmid DNA of V. parahaemolyticus (GenBank accession no. AB972427.1) (Kondo et al. 2014) using PrimerExplorer software version 4 (Fujitsu Limited, https://primerexplorer.jp/lamp4.0.0/index.html). The primer set consisted two outer primers (F3 and B3) and two inner primers (FIP and BIP) and recognized six respective regions of the target sequence (Table 1). To check the specificity of AHPND-LAMP in discriminating between AHPND and non-AHPND strain of V. parahaemolyticus, the target sequences were amplified by the LAMP method (Notomi et al. 2000; Mori et al. 2001) using a Loopamp DNA amplification kit (Eiken Chemical Co., Ltd.). The LAMP reaction mixture contained 12.5 lL of 2 9 reaction mix (Eiken Chemical Correspondence I Hirono, Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo, 108-8477, Japan (e-mail: hirono@kaiyodai.ac.jp)

Identification of novel copper/zinc superoxide dismutase (Cu/ZnSOD) genes in kuruma shrimp Marsupenaeus japonicus

Superoxide dismutases (SODs) protect cells from superoxides, but in invertebrates, they also have role in the innate immune system. In this study, the genes for five isoforms of copper/zinc superoxide dismutase (MjCu/ZnSOD) gene were identified and sequenced in kuruma shrimp, Marsupenaeus japonicus. The coding parts of the genes ranged from 516 to 585 bp in length and encoded from 172 to 194 amino acids. Structure, phylogenetic and BLAST analyses indicated that MjCu/ZnSOD isoform_3 and _5 belonged to extracellular Cu/ZnSOD (ecSOD) group while the other three isoforms belong to the intracellular Cu/ZnSOD family. In healthy shrimp, the highest expressions of isoform 2, 3 and 4 were in the gills, whereas the expression of isoform 5 was highest in hemocytes. Challenging the shrimp with WSSV and Vibrio penaeicida up-regulated the mRNA expressions of isoforms 3 and 5, suggesting that these isoforms have roles in the innate immune system of kuruma shrimp.

Identification of enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri by expressed sequence tag analysis

Expressed sequence tag (EST) analyses were performed with the aim of identifying enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri. Of the 768 ESTs identified and sequenced, 669 were grouped into 324 clusters. Of these clusters, 123 comprising 245 ESTs were found to be homologous to genes reported to date. Among these, 43 clusters were annotated as enzymes according to the Enzyme Commission (EC) numbering system. Two EC groups, oxidoreductases and hydrolases, possessed a large number of ESTs. A cluster homologous to the glutathione peroxidase, an enzyme in the oxidoreductase group, contained 16 ESTs, which accounted for 2.4% of the total ESTs sequenced. There are three serine proteases, three cathepsins, two triacylgricerol lipases, and two chitinases among the clusters homologous to the enzymes in the hydrolase group. Since the squid liver functions in the digestive process, these enzymes would be involved in food digestion. Our data provide information on the various types of enzymes expressed in the squid liver and may provide a useful basis for further characterization of these enzymes.