Program

There is a conclusive evidence that a fish-specific whole genome duplication took place in ray-finned fish around 320 million years ago in addition to two rounds of the genome duplication events early in vertebrate evolution. Furthermore, cyprinid and salmonid fishes appear to be tetraploid because of its chromosome number and its high DNA content. In fish, there have been a number of reports to show the presence of multiple genes, e.g. cytokines: TNFα, IL-1β; lymphocyte cell surface markers: CD4, CD8; compliment components: C2, C3, etc. The additional number of genes resulting from genome or chromosomal duplication might have creative roles in evolution such as speciation, adaptation, diversification, and promotion of new functions, although differential roles of the isoforms have yet to be clarified in most cases. We hypothesize that fish develop different defense strategy from that of mammals by producing diversified isotypes to compensate their immune system being rather simple and undifferentiated compared to that of mammals. In this symposium, we’d like to discuss about the significance of diversified isotypes of immune-related genes focusing on the differential roles of IFNγ isoforms in the immune response of ginbuna crucian carp, Carassius auratus langsdorfii.

Membrane proteins are good basis for classification of different cell types. In crustaceans including shrimps, circulating hemocytes are characterized into three sub-populations based on morphology by microscopic observation. However, the molecular markers like human CD markers have not been reported in shrimp. Therefore, in this study, we identified a membrane protein, integrin alpha subunit (Mj-intg_alpha), as a molecular marker and detected its localization on hemocytes to classify sub-populations.
We produced recombinant partial Mj-intg_alpha (rMj-intg_alpha) protein in Escherichia coli to prepare rabbit antiserum against rMj-intg_alpha. From total antiserum, we purified rabbit IgG. The reactivity of purified IgG against total protein of hemocytes was confirmed by Western-blot analysis. Then, localization and specificity of Mj-intg_alpha against hemocytes was detected by immunostaining and flow cytometry.
By Western-blot analysis, purified IgG reacted against rMj-intg_alpha and 80-90 kDa molecules from hemocytes considered as a light chain of Mj-intg_alpha. By immunostaining, the localization of Mj-intg_alpha was detected around membrane of hemocytes. Additionally, the specificity of Mj-intg_alpha was validated in 20–40 % of granular containing hemocytes by flow cytometry.
Using this antibody, hemocytes were divided into two groups, Mj-intg_alpha+ and - hemocytes. Therefore, this anti-rMj-intg_alpha antibody might pave the way for functional characterization of shrimp hemocytes.

The Pacific oyster, Crassostrea gigas, is one of the most important species in worldwide aquaculture production. In this study, we aimed to examine immune response of C. gigas hemocytes against pathogen-associated molecular patterns (PAMPs; random double-stranded RNA and poly I:C). We quantitatively compared the hemocyte proteome profiles using isobaric tags for relative and absolute quantization (iTRAQ)-coupled 2D LC-MS/MS. The differential proteomes of C. gigas hemocytes were analyzed samples prior to injection with random double-stranded RNA or poly I:C, and at 24 hours post-injection hemocytes. A total of 4798 proteins were identified by LC-MS/MS and sequencing analysis. Using 2.0-fold change in expression as a significant benchmark, 164 (random ds RNA) and 148 (poly I:C) differentially expressed proteins were identified. In the ds RNA-injected group, 82 proteins were up-regulated and 82 proteins were down-regulated. Seventeen differential proteins were involved in immune responses and environmental stress. Up-regulated proteins included three isoforms of C1q-like proteins 4, C1q-TNF-related protein, mollscidin-1 as an antimicrobial peptide and heavy metal-binding protein. Down-regulated proteins included interferon-induced protein L44-like protein, galectin-4, hemagglutinin and HSP 70 protein 12A. Similar results were obtained from the C. gigas hemocytes in the poly I:C-injected group. We consider that his is the first report of iTRAQ-based proteomic response of C. gigas hemocytes against ds RNA injection.

Our group has identified a CD46-like molecule, termed teleost complement regulatory membrane protein or Tecrem, in a few cyprinid fish species and has shown its regulatory function on complement activation at the protein level. In the present study, we have explored a homeostatic role of Tecrem in the maintenance of fish epithelium, by analyzing expression behavior of Tecrem on an epithelial cell line (KF-1) derived from carp fin. Flow cytometric analysis of Tecrem expression on KF-1 using anti-carp Tecrem monoclonal antibody (MAb) (1F12) suggested that Tecrem expression may be affected by cell aggregation and adhesion. Fluorescent microscopic observation and an ELISA-based assay also indicated a role of Tecrem in the adhesion of KF-1 to the surface of culture media. Furthermore, 1F12 MAb deposited on the culture plate significantly enhanced an early stage of cell adhesion process of KF-1.

We have also prepared recombinant Tecrem proteins in the bacterial expression system for functional analysis of Tecrem. Among the four short consensus repeat (SCR) modules making up the extracellular domains of Tecrem, the N-terminal two SCRs (rSCR1-2) and the C-terminal two SCRs (rSCR3-4) were separately expressed as 6xHis-tagged soluble protein using pCold-I vector and Origami B strain. Cell adhesion and wound-healing assays done on KF-1 cells showed that the cell adhesion and wound healing is enhanced when the cells were incubated with anti-SCR1-2 and anti-SCR3-4 polyclonal antibodies and with 1F12 MAb. Increased cell proliferation and wound healing of KF-1 cells upon cTecrem activation suggests that the normal functional role of cTecrem on epithelial cells may rather be homeostatic via the induction of surface barrier repair upon pathogen entry or any other injury. Cell signalling mechanisms to mediate the Tecrem-induced epitherial cell reseponse are currently under investigation.

Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which have been considered a functional equivalent of mammalian platelets. In addition to their hemostatic effects, thrombocytes also work as a component of the immune system, including phagocytosis. In an early stage of viral infection, several viral-specific molecular patterns including single/double-stranded RNA (ss/dsRNA) are recognized by innate pattern recognition receptors, such as Toll-like receptors (TLRs), thereby production of type I interferon (IFN) is induced. In the present study, we report sensitivity to synthetic viral molecule mimicries of common carp (Cyprinus carpio) thrombocytes, as the natural type I IFN producing cells. Magnetic-sorted mAb⁺ thrombocytes and mAb^- peripheral blood leukocytes (PBLs) were incubated with polyinosinic:polycytidylic acid (poly(I:C); an analog of dsRNA recognized by TLR3 in mammals) or resiquimod (R-848, an agonist of TLR7/8, which detect ssRNA). qPCR analysis showed that thrombocytes had a potential to produce large amount of type I IFN, more than ten-fold higher than that of the PBL fraction. R-848 acutely activated thrombocytes, followed by the upregulation of the IFN production, whereas increase of the IFN expression by poly(I:C) was detected only in the PBL fraction, suggesting that thrombocytes recognize ssRNA but not poly(I:C) or dsRNA. Despite the insensitivity to poly(I:C), however, thrombocytes expressed TLR3s (TLR3.1/3.2) in a level comparable with that in other leukocytes. Meanwhile, TLR22, a fish-specific TLR, which also senses dsRNA, was not expressed in the thrombocyte fractions, suggesting that the poly(I:C), or maybe dsRNA, was not recognized by carp TLR3 but by TLR22. Those pattern-recognition characteristics and type I IFN producing capacity of carp thrombocytes are analogous to that of the mammalian plasmacytoid dendritic cells, indicating that thrombocytes are important immune components against viral infections for fish and may be a target for the strategy of vaccine development.

We previously reported that Aeromonas salmonicida subsp. salmonicida (A.s.s.) bacterin is taken up by gill epithelial antigen sampling (GAS) cells. GAS cells are typically recognized by the lectin Ulex europaeus agglutinin (UEA)-1 same as mammalian M cells. In this study, we performed a comprehensive gene expression analysis to identify genes typically expressed in GAS cells.
A.s.s. were cultured, inactivated with 0.3% formalin. Rainbow trout were bath-vaccinated with SYTO61-stained A.s.s. bacterin at a concentration of 10⁷CFU/ml. Epithelial cells were isolated from the gills, stained with FITC-labeled UEA-1 and subjected to flow cytometry, followed by flow sorting. Two cell populations, A.s.s.⁺ UEA-1⁺ (GAS cells) and A.s.s.⁺ UEA-1^- were isolated and total RNA was extracted. The cDNA library of each cell population was sequenced by a next generation sequencer, Miseq. Gene expression analysis based on read counts was performed using Trinity assemble software and several genes of interest were validated by real-time PCR.
Flow cytometry revealed that two cell populations taken up A.s.s. bacterin in the gill epithelium: GAS cells and A.s.s.⁺ UEA-1^- (single positive, SP) cells. The percentage of each cell group was 22% in GAS cells, and 5% in the SP cell population. These two populations showed different characteristics in flow cytometry according to their forward and side scatters. GAS cells highly expressed Annexin A5 which is typically expressed in mammalian M cells, the interleukin (IL)-17 receptor and claudin that are typically in mammalian epithelial cells. In addition, a membrane protein gene, zymogen granule membrane 16-like (ZG16) was expressed in GAS cells at high levels. In contrast, SP cells highly expressed CD83 and IL-12 that are typical for mammalian macrophages/monocytes. These data indicate that Annexin A5 and ZG16 are marker genes of GAS cells.

Rock bream (Oplegnathus faciatus) farms in Korea have been suffering from rock bream iridovirus (RBIV) infection every year. In this study, we tried to understand physiological responses occurring in rock bream infected with RBIV through co-expression and DEG analysis using spleen RNA-seq data. There were four groups depending on infection intensity; 0 week-control (0C, 3.54 ± 2.07 x 101 copies / mg), 0 week-heavy infection (0H, 6.48 ± 6.79 × 107 copies / mg), 3 week-light infection (3L, 2.08 ± 2.07 x 103 copies / mg) and 3 week-control (3C, under detection limit). The spleen samples (n=5/group) were taken from each group at week 0 and 3. From iso-seq (PacBio) data, 68,211 unigenes were produced and used as reference, and thus approximately 85% of reads generated by Illumina Hiseq 2500 were mapped onto the reference. Co-expression analysis was performed using the WGCNA (weighted gene co-expression network analysis) package with 37210 unigenes (total read counts < 10 per group). Significant modules (p-value <0.05, |correlation with viral copies| >0.5) were selected and analyzed using KEGG and GO. WGCNA revealed 26 modules that were assigned unique colors, and each co-expression module consisted of genes exhibiting similar expression patterns. In blue (positive, 10098 genes) module, genes in Group 0H were up-regulated in pathways related to cancer, HTLV-I infection, RNA transport, and protein processing in endoplasmic reticulum compared with other groups. This shows rock bream infected with RBIV elicit defense responses,, and increase mRNA production in nucleus and export to the cytoplasm and processing of proteins. In Group 0H, expression of genes associated with the immune and inflammatory responses, MHC class II-related, and chemotaxis was down-regulated, but genes related to interferon and Mx, and immunoglobulin heavy chain were up-regulated. This transcriptome approach will facilitate new insights into understanding of global rock bream-RBIV interactions.