Keiichiro Koiwai

Extracellular trap formation in kuruma shrimp (Marsupenaeus japonicus) hemocytes is coupled with c-type lysozyme.

In invertebrates, hemocytes play an important role in immune responses. Recently, a novel form of innate immune mechanism called extracellular traps (ETs) was identified in shrimps, where DNA and antimicrobial peptides form complex structure to entrap the invading microbes. In this study, we detected the formation of ETs from hemocytes of kuruma shrimp in response to various stimulations, including phorbol myristate acetate (PMA), lipopolysaccharide (LPS), peptidoglycan (PGN) and Escherichia coli. E. coli cells were also found to be trapped by ET fibers. Fluorescence imaging revealed that c-type lysozyme proteins were released around the ET complex after E. coli stimulation, suggesting the presence of a coupled antimicrobial immune response involving ET formation and AMP release.

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)

Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation.

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

ABSTRACT 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. HighlightsA viral responsive protein of M. japonicus was cloned and characterized.MjVRP is mainly expressed in haemocytes and located close to the nucleus of semi‐granular and granular cells.MjVRP mRNA expression level is increased after WSSV challenge.The number of positive haemocytes was increased after WSSV challenge.

A rapid method for simultaneously diagnosing four shrimp diseases using PCR-DNA chromatography method

Shrimp farming accounted for more than half of world production of shrimp in 2014 (Food and Agricultural Organization, 2017a). In shrimp farming, viral, bacterial, microspordium and fungal diseases cause severe losses, so rapid methods for detecting them are needed. Polymerase chain reaction (PCR)-based detection method is widely used as screening for many pathogenic bacteria due to its convenience and sensitivity. Common viral diseases include white spot disease (WSD) and infectious hypodermal and haematopoietic necrosis (IHHN). A common bacterial disease is acute hepatopancreas necrosis disease (AHPND). Conventional PCR detection methods targeting the agents that cause these diseases are recommended (World Organisation for Animal Health, 2016). Recently, Enterocytozoon hepatopenaei (EHP) was identified as the microsporidium that causes growth retardation in farmed shrimp (Tangprasittipap et al., 2013). Conventional PCR has also been used to detect EHP infection (Tang et al., 2015; Tangprasittipap et al., 2013). Conventional PCR requires agarose/acrylamide gel electrophoresis and a device for visualizing the gel. Another method for detecting the PCR product is single-strand tag hybridization (STH) chromatographic printed array strip (PAS) method (Monden et al., 2014; Ohshiro, Miyagi, Tamaki, Mizuno, & Ezaki, 2016; Tian et al., 2014). In this method, multiplex PCR products can be visualized with high sensitivity within 15 min. Moreover, these chromatography strips are portable and ideal for field testing, as it eliminates the need for an electrophoresis equipment and preparation of gels that needs also require gel documentation equipment for viewing the PCR results. The method consists of a multiplex PCR in which multiple primer sets are tagged with a specific linker and biotin. After obtaining the PCR products, the PCR products then rise by capillary action in a tiny DNA chromatography strip to a point determined and hybridized by tag linker, stained with streptavidin-coated blue latex, which is visible to the naked eye (Figure 1). In this study, we developed a detection system for four shrimp diseases (WSD, IHHN, AHPND and EHP infections) using multiplex PCR and STH chromatographic PAS, named PCR-DNA chromatography. A total of 89 shrimp DNA samples of Penaeus monodon and Litopenaeus vannamei were collected in Thailand for this study. Total genomic DNA of these samples was extracted using taco Nucleic Acid Automatic Extraction System (GeneReach Biotechnology Corp, Taichung City, Taiwan). For each of the DNA samples, each disease was detected by conventional PCR using KAPA2G Fast Multiplex Mix (Kapa Biosystems, Wilmington, MA, USA) or KAPA 2G Fast Hot Start Ready Mix with dye (Kapa Biosystems) referring to previous reports: WSD (Lo et al., 1996), IHHN (Tang, Navarro, & Lightner, 2007), AHPND (Tinwongger et al., 2014) and EHP infections (Tangprasittipap et al., 2013). We obtained 30 samples that were positive for white spot virus (WSV) which is the causative virus of WSD, seven samples that were positive for infectious hypodermal and haematopoietic necrosis virus (IHHNV) which is the causative virus of IHHN, 14 samples that were positive for toxin gene of a virulent strain of Vibrio parahaemolyticus of AHPND, 19 samples that were positive for EHP, 14 samples that were double-positive for IHHNV and EHP and samples that were negative for each of these diseases. To develop PCR-DNA chromatography, we targeted WSD, IHHN, AHPND and EHP infections and 16S rRNA/tRNA/12S rRNA mitochondrial region (an internal control) using primers previously reported for WSD (Kiatpathomchai et al., 2005), IHHN (Tang et al., 2007), AHPND (Tinwongger et al., 2014), EHP infections (Tangprasittipap et al., 2013) and 16S rRNA/tRNA/12S rRNA (Pascoal, Barros-Vel azquez, Cepeda, Gallardo, & Calo-Mata, 2008) *These authors contributed equally to this work. Received: 9 July 2017 | Revised: 22 August 2017 | Accepted: 23 August 2017 DOI: 10.1111/jfd.12732

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

ABSTRACT 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. HighlightsFull‐length MjVIG1 cDNA was identified in Marsupenaeus japonicus hemocytes.MjVIG1 transcripts significantly increased after WSSV infection but not after V. parahaemolyticus and YHV infections.The ratio of MjVIG1+ hemocytes significantly increased and MjVIG1+ hemocytes number slightly increased after WSSV infection.MjVIG1 might be a WSSV‐specific induced gene in M. japonicus hemocytes.

Identification of endonuclease domain-containing 1 gene in Japanese flounder Paralichthys olivaceus.

The mRNA level of the endonuclease domain-containing 1 gene (Jf_ENDOD1) in Japanese flounder Paralichthys olivaceus kidney was significantly increased after injection of formalin-killed bacteria cells (FKC) in the previous microarray study. ENDOD1 is a member of the DNA/RNA non-specific nucleases family, and its role in fish immunity has not been reported. The open reading frame of Jf_ENDOD1 cDNA was 912 bp, encoding 303 amino acids. The first 27 amino acids were predicted to be a signal peptide and the mature Jf_ENDOD1 was calculated as 32 kDa. The amino acid sequence of Jf_ENDOD1 showed 76% identity to that of large yellow croaker Larimichthys crocea. Transcripts of Jf_ENDOD1 were marginally detected in all sampled tissues from healthy fish, while they were significantly detected in brain, kidney, spleen and intestine at 6 h post FKC injection. Jf_ENDOD1 recombinant protein produced in Escherichia coli showed DNase activity. Furthermore, to evaluate the DNase activities in vivo, total proteins from Japanese flounder kidney and spleen were extracted at 12, 24 and 72 h post Edwardsiella tarda FKC injection. The DNase activity of extracted protein was higher in treated fish than in untreated fish. Since the mRNA levels were significantly up-regulated after the FKC treatment, Jf_ENDOD1 might be responsible for the activities.

Development of 11 Ecklonia radicosa (Phaeophyceae, Laminariales) SSRs markers using next-generation sequencing and intra-genus amplification analysis

Polymorphic simple sequence repeat (SSR) markers of Ecklonia radicosa were developed using Illumina MiSeq next-generation sequencing (NGS) analysis. In total, 3112 SSRs (di- to hexanucleotide motifs repeated more than six times) were identified from 61.5-Mb assembled genomic DNA using MISA perl script. Among the SSRs, di- (853 loci, 27.4%) and trinucleotides (1813 loci, 58.3%) were dominant, while tetra- (172 loci, 5.5%), penta- (175 loci, 5.6%), and hexanucleotides (99 loci, 3.2%) were not common. After specific amplification and polymorphic tests of 75 selected loci (tri- to hexanucleotide motifs repeated more than eight times), 11 SSR markers (Eradic01–11) were successfully developed. The range of the number of alleles, observed heterozygosity, and expected heterozygosity in the 11 markers was 3–13, 0.200–0.683, and 0.258–0.864, respectively. Polymorphic information content (PIC) analysis indicated that eight markers (Eradic01, 02, 04, 05, 06, 08, 09, and 11) were highly informative (PIC > 0.5) and the other three were reasonably informative (0.5 < PIC < 0.25). In addition, intra-genus amplification was obtained in all markers except for Eradic02, 08, and 11. These markers could help to reveal the genetic diversity and population structure of E. radicosa.

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

ABSTRACT 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. HIGHLIGHTSSessile hemocytes of gills, heart and lymphoid organs were enriched.Transcriptome analysis between circulating and sessile hemocytes was conducted.Sessile hemocytes may have a different type of hemocytes from circulating hemocytes.