Kyushu University Academic Staff Educational and Research Activities Database
Researcher information (To researchers) Need Help? How to update
Miki Nakao Last modified date:2022.11.04

Professor / Division of Molecular Bioscience
Department of Bioscience and Biotechnology
Faculty of Agriculture


Graduate School
Undergraduate School
Administration Post
Dean of the School of Agriculture
Dean of the Graduate School of Bioresource and Bioenvironmental Sciences
Dean of the Faculty of Agriculture


E-Mail *Since the e-mail address is not displayed in Internet Explorer, please use another web browser:Google Chrome, safari.
Homepage
https://kyushu-u.pure.elsevier.com/en/persons/miki-nakao
 Reseacher Profiling Tool Kyushu University Pure
http://mikimnakaojapan.blogspot.com/
Miki's Kernel & Shell (A personal diary on my academic and non-academic activities) .
http://www.agr.kyushu-u.ac.jp/biosci-biotech/suizoku/
Phone
092-802-4791
Fax
092-802-4791
Academic Degree
PhD
Country of degree conferring institution (Overseas)
No
Field of Specialization
Marine Biochemistry, Comparative Immunology, Immunogenetics, Molecular Immunology
Total Priod of education and research career in the foreign country
00years04months
Outline Activities
My major research interest is structural and functional diversity of the complement components in bony fish. In contrast to mammalian complement system, bony fish have multiple copies of the genes encoding several complement components such as C3, C4, C5 and factor B/C2. Currently I am analyzing biological and evolutionary impact of the complement diversity on the innate immunity of the bony fish. Data obtained will be utilized for establishment of prevention of infectious diseases, which damage many kinds of cultured fish stock, and also for genotype-based selection of fish lineage with higher degree of host defense. Other research subject of my interest is phylogeny of the innate and adaptive immune system. Molecular cloning of innate immune components such as cytokines, complement components, and lectins will give some insights into genetic (genomic) mechanism that generated a number of immune-related genes in the lower vertebrates and invertebrates. Application of the finding obtained by the studies will be of importance to develop better means of disease prevention in fish and invertebrate aquaculture.
Research
Research Interests
  • Elucidation of structure, function and their diversity of the complement system in fish
    keyword : fish, complement, structure, function, diversity
    1993.04Identification of genes encoding zebrafish complement components..
  • Comparative Immunology of innate immune factors in aquatic vertebrates and invertebrates
    keyword : fish, aquatic animals, invertebrates, comparative immunology, innate immunity
    2000.04Molecular and cellular biological studies on the immune system of fish, using cyprinid species (common carp, ginbuna crucian carp, and zebrafish).
  • Elucidation of crosstalk between innate and adaptive immune systems in fish
    keyword : fish, innate immunity, adaptive immunity, acquired immunity, cellular immunity, interaction, crosstalk
    2004.04Structural and functional diversity of the genes encoding complement components in the common carp..
Academic Activities
Books
1. Miki Nakao, Tomonori Somamoto, The evolution of complement system functions and pathways in vertebrates. in THE EVOLUTION OF THE IMMUNE SYSTEM: A BALANCE BETWEEN CONSERVATION AND DIVERSITY, Elsevier, in press, 2015.08, An ancestral complement system is considered to be an opsonic system that seems a combination of the lectin and alternative pathways known in mammalian complement. The ancestral system equipped with lectin-like pattern-recognition molecule linked with specific proteases homologous to MASP and factor B, which cleave C3 into physiologically active forms with inflammatory and opsonic functions, as seen in primitive invertebrate species. In vertebrates, two rounds of whole-genome duplication have generated two additional reaction cascades, the classical and lytic pathway, providing jawed vertebrates with much more specific and efficient ability of pathogen elimination. A trace of molecular and functional evidence that represent a transient status from invertebrate prototypic complement to full equipped system can be found in extant jawless species such as lamprey. A striking diversity of complement component isoforms unique to teleost fish is also discussed in a functional context..
2. Evolution of complement system in vertebrates in "Introduction to Complement", Ed. by Ooi H, Kinoshita T, and Matsushita M..
Reports
1. Multifaceted roles of the complement system: an evolutionary implication.
2. Evolution of the complement system.
3. Evolution and phylogeny of the complement system and complementology.
4. Van Muiswinkel WB, Miki Nakao, A short history of research on immunity to infectious diseases in fish, Dev Comp Immunol. , doi: 10.1016/j.dci.2013.08.016, 2013.08, This review describes the history of research on immunity to infectious diseases of fish in the period between 1965 and today. Special attention is paid to those studies, which are dealing with the interaction between immune system and invading pathogens in bony fish. Moreover, additional biographic information will be provided of people involved. In the 1960s and 1970s the focus of most studies was on humoral (Ig, B-cell) responses. Thorough studies on specific cellular (T-cell) responses and innate immunity (lectins, lysozyme, interferon, phagocytic cells) became available later. In the period between 1980 and today an overwhelming amount of data on regulation (e.g. cell cooperation, cytokines) and cell surface receptors (e.g. T-cell receptor; MHC) was published. It became also clear, that innate responses were often interacting with the acquired immune responses. Fish turned out to be vertebrates like all others with a sophisticated immune system showing specificity and memory. These basic data on the immune system could be applied in vaccination or in selection of disease resistant fish. Successful vaccines against bacterial diseases became available in the 1970s and 1980s. Effective anti-viral vaccines appeared from the 1980s onwards. There is no doubt, that Fish Immunology has become a flourishing science by the end of the 20th century and has contributed to our understanding of fish diseases as well as the success of aquaculture..
5. Nakao M, Tsujikura M, Ichiki S, Vo TK, Somamoto T., The complement system in teleost fish: Progress of post-homolog-hunting researches., Dev Comp Immunol. , Epub, 2011.05.
6. Phylogeny and evolution of activation pathways of the complement system.
7. Nakao M, Kato-Unoki Y, Nakahara M, Mutsuro J and Somamoto T, Diversified components of bony fish complement system: More genes for robuster innate defense?, Advances in Experimental Medicine and Biology, 586: 121-138, 2006.03.
8. Nakao M, Mutsuro J, Nakahara M, Kato Y, Yano T, Expansion of genes encoding complement components in bony fish: biological implications of the complement diversity., Developmental and Comparative Immunology, 27: 749-762., 2003.01.
Papers
1. Prakash H, Motobe S, Nagasawa T, Somamoto T, Nakao M, Homeostatic Functions of Tecrem, a CD46-Like Regulatory Protein of Complement Activation, on Epithelial Cells in Carp Fish, Journal of Marine Science and Engineering, https://doi.org/10.3390/jmse9070687, 97, 7, 687, 2021.06, Fish mucosal surface is a significant interface for pathogens to infect from an aqueous environment. In addition to mucosal innate and adaptive immune factors, epithelial cells are considered as a significant physical barrier against microbial invasion. Previously, we identified a mammalian CD46-like complement regulatory protein (Tecrem) in teleost and detected its expression on epithelial cells derived from fin, suggesting its physiological role on the fish surface. This study examines the homeostatic roles of Tecrem in maintaining the fish epithelium, by analyzing the expression behavior of Tecrem on the fin-derived epithelial cell lines (KF-1 from the common carp and CFS from ginbuna crucian carp) using monoclonal and polyclonal anti-Tecrem antibodies. Expression of KF-1 protein was associated with the adhesion of KF-1, and the adhesion was enhanced by anti-Tecrem treatments of the cells. Stimulation of the epithelial cells with anti-Tecrem enhanced wound healing, protein expression of tight-junction proteins, and cell density of the KF-1 and CFS monolayer culture. These results suggest that Tecrem on epithelial cells play a homeostatic role in maintaining intactness of the surface epithelial barrier, implying that modification of Tecrem expression may develop a novel tool to improve the first-line defense against pathogens in aquaculture..
2. Yumie Tokunaga, Masamichi Shirouzu, Ryota Sugahara, Yasutoshi Yoshiura, Ikunari Kiryu, Mitsuru Ototake, Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao, Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish
Implication for the robust innate defense mechanisms of teleosts, Scientific Reports, 10.1038/s41598-017-08000-2, 7, 1, 2017.12, rag1 -/- zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1 -/- zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1 -/- fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1 -/- fish possess innate protection equivalent to that of rag1 -/- fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models..
3. Kolder IC, van der Plas-Duivesteijn SJ, Tan G, Wiegertjes GF, Forlenza M, Guler AT, Travin DY, Miki Nakao, Moritomo T, Irnazarow I, den Dunnen JT, Anvar SY, Jansen HJ, Dirks RP, Palmblad M, Lenhard B, Henkel CV, Spaink HP, A full-body transcriptome and proteome resource for the European common carp., BMC Genomics, 10.1186/s12864-016-3038-y, 17, 701-1-701-12, 2016.09, [URL], BACKGROUND:
The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies.
RESULTS:
Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data.
CONCLUSIONS:
The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes..
4. Nur I, Abdelkhalek NK, Motobe S, Nakamura R, Tsujikura M, Tomonori Somamoto, Miki Nakao, Functional analysis of membrane-bound complement regulatory protein on T-cell immune response in ginbuna crucian carp, Molecular Immunology, 10.1016/j.molimm.2015.11.010, 70, 1-7, 2016.02, [URL], Complements have long been considered to be a pivotal component in innate immunity. Recent researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity. Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly identified in common carp and shown to function as a complement regulator. However, it remains unclear whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecipitation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed on blood cells including CD8α(+) and CD4(+) lymphocytes. gTecrem expression on leucocyte surface was enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the anti-Tecrem mAb significantly inhibited the proliferative activity of PHA-stimulated peripheral blood lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction pathway for T-cell activation. These findings indicate that Tecrem may act as a T-cell moderator and imply that the complement system in teleost, as well as mammals, plays an important role for linking adaptive and innate immunity..
5. Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao, Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes, Developmental and Comparative Immunology, 10.1016/j.dci.2015.05.002, 52, 2, 107-111, 2015.07, Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb+ thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb− leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb− leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair..
6. Tomonori Somamoto, Yuhei Miura, Teruyuki Nakanishi, Miki Nakao, Local and systemic adaptive immune responses toward viral infection via gills in ginbuna crucian carp, Developmental and Comparative Immunology, 10.1016/j.dci.2015.04.016, 52, 1, 81-87, 2015.07, Recent studies on fish immunity highlighted the significance of gills as mucosal immune tissues. To understand potential of gills as vaccination sites for inducing adaptive systemic immunity, we investigated virus-specific cell-mediated and humoral immune responses following a “per-gill infection method”, which directly exposes virus only to gills. The viral load in crucian carp hematopoietic necrosis virus (CHNV)-infected gills decreased after peaking at a particular time point. Furthermore, the viral titers in the gills following the secondary infection were lower than that after the primary infection, indicating that local adaptive immunity helped the elimination of virus. Gene expression analysis demonstrated that IFN-γ in gills and perforin in kidney were increased after the gill infection. CD8+ cells in kidney leukocytes increased after the secondary infection, whereas IgM+ cells decreased. These results suggest that IFN-γ and CTL contribute in controlling CHNV-replication in gills and kidney. Gill infection could induce specific cell-mediated cytotoxicity of peripheral blood leukocytes (PBL) and secretion of CHNV-specific IgM in serum, indicating that local priming of the gill site can generate adaptive systemic immunity. Thus, the gills could be prospective antigen-sensitization sites for mucosal vaccination..
7. Masakazu Tsujikura, Takahiro Nagasawa, Satoko Ichiki, Ryota Nakamura, Tomonori Somamoto, Miki Nakao, A CD46-like Molecule Functional in Teleost Fish Represents an Ancestral Form of Membrane-Bound Regulators of Complement Activation, JOURNAL OF IMMUNOLOGY, 10.4049/jimmunol.1303179, 194, 1, 262-272, 2015.01, In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors. Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a membrane-bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized for its complement-regulatory roles. Carp Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus, as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2 RCA in bony fish, implying the host-cell protection is an evolutionarily conserved mechanism in regulation of the complement system..
8. Takahiro Nagasawa, Chihaya Nakayasu, Aja M. Rieger, Daniel R. Barreda, Tomonori Somamoto, Miki Nakao, Phagocytosis by thrombocytes is a conserved innate immune mechanism in lower vertebrates. , Frontiers in Immunology, doi: 10.3389/fimmu.2014.00445, 5, 445, Article 445, 2014.09, Thrombocytes, nucleated hemostatic blood cells of non-mammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5-3 μm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions..
9. Abdel-Salam, Soha G. R., Masakazu Kondo, Masakazu Tsujikura, Tomonori Somamoto, Miki Nakao, Purification and functional characterization of complement C3 and a novel zymosan-binding protein in tilapia serum, Fisheries Science, 10.1007/s12562-014-0700-7, 80, 2, 301-310, 2014.03, Zymosan, a yeast cell wall preparation that binds activated forms of complement C3, is a useful model target to activate the complement system. In our trial to analyze C3 diversity in Nile tilapia at the protein level using zymosan, we found that a novel 240-kDa serum protein (ZBP-240) also bound to zymosan in addition to C3-derived fragments. In the present study, we aimed to characterize tilapia C3 and ZBP-240, focusing on their immune-related functions. Four distinct C3 isoforms were purified from tilapia serum and shown to possess an intrachain thioester bond. ZBP-240 was also isolated from tilapia serum and examined for its binding properties to various microbial targets. As a result, ZBP-240 showed a wide spectrum of binding to Gram-positive and Gram-negative bacteria and yeasts. Amino acid sequence analysis of CNBr fragments of ZBP-240 suggested that this is a novel protein with no homologous sequence in protein databases. It was also suggested that the binding of ZBP-240 to microbes largely depends on hydrophobic interactions in a divalent-cation-independent manner, and that there may be a divalent-cation-dependent factor that enhances the binding of ZBP-240 in tilapia serum. Interestingly, ZBP-240 showed opsonic activity for tilapia kidney phagocytes at a level comparable to that of C3, implying that ZBP-240 is a novel teleost opsonic serum protein..
10. Indriyani Nur, Hikari Harada, Masakazu Tsujikura, Tomonori Somamoto, Miki Nakao, Molecular characterization and expression analysis of three membrane-bound complement regulatory protein isoforms in the ginbuna crucian carp Carassius auratus langsdorfii., Fish Shellfish Immunol., 10.1016/j.fsi.2013.08.002, 35, 4, 1333-1337, 2013.10, Regulators of complement activation (RCA) play a role in protecting cells from excessive complement activation in humans. cDNA corresponding to three isoforms of teleost membrane-bound RCA protein (gTecrem) have been identified in the ginbuna crucian carp. gTecrem-1 consists of seven short consensus repeats (SCRs), whereas gTecrem-2 and gTecrem-3 have four SCRs. While gTecrem-1 possesses a tyrosine phosphorylation site in its cytoplasmic region, gTecrem-2 and gTecrem-3 lack the site. Tissue distribution analysis showed that gTecrem-1 and gTecrem-2 mRNAs were expressed in almost all tissues examined, whereas gTecrem-2 expression was not significantly detected in gill, liver, or intestine. Furthermore, analysis showed that gTecrem-1 was expressed in both peripheral blood leukocytes (PBLs) and erythrocytes and was also expressed in T cell subsets such as CD4+, CD8+ T cells, and IgM+ B cells. gTecrem-2 expression was not detected in either PBLs or erythrocytes, whereas gTecrem-3 was expressed only in erythrocytes. These results suggested that gTecrem isoforms may serve different functional roles; gTecrem-1, which is expressed in T cells and possesses a tyrosine phosphorylation site, may act as a complement regulator and a cellular receptor in adaptive immunity..
11. Ichiki S, Kato-Unoki Y, Somamoto T, Nakao M., The binding spectra of carp C3 isotypes against natural targets independent of the binding
specificity of their thioester., Developmental and Comparative Immunology, 10.1016/j.dci.2012.03.004., 38, 1, 10-16, 2012.08, The central component of complement, C3, plays a versatile role in innate immune defense of vertebrates and some invertebrates. A notable molecular characteristic of this component is an intra-chain thioester site that enables C3 to bind covalently to its target. It has been reported that the binding preference of the thioester to hydroxyl or amino groups is primarily defined by presence or absence of the catalytic histidine residue at position 1126 in human C3. In teleosts, a unique C3 (non-His type) has been found, in addition to the common His type C3. These distinct C3 isoforms may provide diversity in the target-binding attributable to the different binding specificities of their thioesters. In the present study, we examine the hypothesized correlation of the catalytic histidine with the binding spectra of two major C3 isotypes of carp towards various model and natural targets. The results reveal that non-His type C3, rather than His type C3, has a wider range of binding spectrum, despite the binding specificity of its thioester being limited to amino groups. It is therefore hypothesized that the binding spectra of C3 isotypes are not defined by the binding specificity of the thioester but is more affected by differences in microbe-associated molecular patterns that activate complement. Overall, the present data imply that non-His type C3 plays a significant role against bacterial infections in the fish defense system..
12. VO Kha Tam, Masakazu TSUJIKURA, Tomonori SOMAMOTO, and Miki NAKAO, Identification of cDNA sequences encoding the complement components of zebrafish (Danio rerio), Journal of Faculty of Agriculture, Kyushu University, 54, 2, 373-387, 2009.10.
13. VO Kha Tam, Masakazu TSUJIKURA, Tomonori SOMAMOTO, and Miki NAKAO, Expression responses of the complement components in zebrafish organs after stimulation with poly I:C, mimicry of viral infection, Journal of Faculty of Agriculture, Kyushu University, 54, 2, 389-395, 2009.10.
14. Forlenza M, Nakao M, Wibowo I, Joerink M, Arts JA, Savelkoul HF, Wiegertjes GF, Nitric oxide hinders antibody clearance from the surface of Trypanoplasma borreli and increases susceptibility to complement-mediated lysis., Molecular Immunology, 10.1016/j.molimm.2009.08.011, 46, 16, 3188-3197, 2009.10, Trypanoplasma borreli is an extracellular blood parasite of carp belonging to the same Order (Kinetoplastida) as African trypanosomes. These mammalian parasites have developed different strategies to evade the host immune system including antigenic variation, immunosuppression and clearance of surface-bound antibodies. The latter mechanism allows trypanosomes to use their swimming movement to cause surface-bound antibodies to 'sail' and accumulate at the posterior end of the parasite, to be internalized via the flagellar pocket and be degraded. There is no evidence that T. borreli shows antigenic variation, but during the late phases of infection NO-mediated immunosuppression is observed. High levels of nitric oxide (NO) lead to extensive tissue nitration whereas the parasite itself is not affected. Therefore, the induction of NO has thus far been considered a parasite-driven response with immunosuppressive effects. In the present study, we show that the induction of NO, particularly during the early phase of T. borreli infections, should be re-considered an effective part of the host immune response. We show that T. borreli rapidly removes surface-bound IgM. In addition, moderate concentrations of NO, by hindering surface antibody clearance, maintain high the concentrations of membrane-bound IgM, thereby favoring antibody-dependent complement-mediated parasite lysis. We performed a comprehensive quantitative gene expression analysis of in total seven different complement factors involved in all three activation pathways, differentiating between 1 and 4 isoforms for each complement gene. Our gene expression analysis supports an important role for antibody-dependent complement-mediated lysis of T. borreli in vivo. To our knowledge, NO-dependent inhibition of antibody clearance from the surface of kinetoplastid parasites has not been investigated. Our data support a role for NO as an important player in host-parasite interactions, not only as immune suppressor (late response) but also as immune effector (early response) in infections with bloodstream parasites such as T. borreli..
15. Nevien K. Abdelkhalek, Asuka Komiya, Yoko Kato-Unoki, Tomonori Somamoto, Miki Nakao, Molecular evidence for the existence of two distinct IL-8 lineages of teleost CXC-chemokines, Fish & Shellfish Immunology, 10.1016/j.fsi.2009.08.004, 27, 6, 763-767, 2009.09, Interleukin-8 (IL-8) is a CXC-type chemokine with a chemotactic activity mainly on neutrophils and plays a key role in promoting inflammation. In teleosts, several CXC-chemokines have been cloned and characterized as being IL-8-like. Phylogenetic data however indicate that the reported teleost IL-8-like chemokines are substantially remote from mammalian IL-8, forming a fish-specific clade of IL-8-like chemokines distinct from that of tetrapod IL-8. In the present study, a novel IL-8-like chemokine, designated CaIL-8, has been found in the expressed sequence tags of carp gills and identified as an orthologue of mammalian IL-8. The CaIL-8 transcript encodes 99 amino acids containing a typical CXC motif but lacks an ELR motif, as in most teleost IL-8-like chemokines. Phylogenetic tree constructed by the maximum likelihood method suggests a closer relationship of CaIL-8 with mammalian IL-8 than with other teleost CXC-chemokines reported to be IL-8-like. In a normal unstimulated carp, CaIL-8 mRNA was detected by RT-PCR only in gills, kidney, spleen, heart and peripheral blood leukocytes, in contrast to a previously reported carp IL-8-like chemokine CXCa, which shows ubiquitous basal expression. The results, taken together, are strongly indicative of the presence of two major IL-8-like lineages of CXC-chemokines in teleost..
16. Dong-Ho Shin, Barbara M. Webb, Miki Nakao, Sylvia L. Smith, Characterization of shark complement factor I gene(s): Genomic analysis of a novel shark-specific sequence, Molecular Immunology, 10.1016/j.molimm.2009.04.002, 46, 11-12, 2299-2308, 2009.05, Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and -d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (≤) amino acid identities with each other, 35.4-39.6% and 62.8-65.9% with factor I of mammals and banded houndshark (Triakis scyllium), respectively. The modular structure of the GcIf is similar to that of mammals with one notable exception, the presence of a novel shark-specific sequence between the leader peptide (LP) and the factor I membrane attack complex (FIMAC) domain. The cDNA sequences differ only in the size and composition of the shark-specific region (SSR). Sequence analysis of each SSR has identified within the region two novel short sequences (SS1 and SS2) and three repeat sequences (RS1-3). Genomic analysis has revealed the existence of three introns between the leader peptide and the FIMAC domain, tentatively designated intron 1, intron 2, and intron 3 which span 4067, 2293 and 2082 bp, respectively. Southern blot analysis suggests the presence of a single gene copy for each cDNA type. Phylogenetic analysis suggests that complement factor I of cartilaginous fish diverged prior to the emergence of mammals. All four GcIf cDNA species are expressed in four different tissues and the liver is the main tissue in which expression level of all four is high. This suggests that the expression of GcIf isotypes is tissue-dependent..
17. Nomiyama H, Hieshima K, Osada N, Kato-Unoki Y, Otsuka-Ono K, Takegawa S, Izawa T, Yoshizawa A, Kikuchi Y, Tanase S, Miura R, Kusada J, Nakao M, Yoshie O, Extensive Expansion and Diversification of the Chemokine Gene Family in Zebrafish: Identification of a Novel Chemokine Subfamily CX, BMC Genomics, 10.1186/1471-2164-9-222, 9, Article No. 222, 2008.05, Background: The chemokine family plays important roles in cell migration and activation. In humans, at least 44 members are known. Based on the arrangement of the four conserved cysteine residues, chemokines are now classified into four subfamilies, CXC, CC, XC and CX3C. Given that zebrafish is an important experimental model and teleost fishes constitute an evolutionarily diverse group that forms half the vertebrate species, it would be useful to compare the zebrafish chemokine system with those of mammals. Prior to this study, however, only incomplete lists of the zebrafish chemokine genes were reported. Results: We systematically searched chemokine genes in the zebrafish genome and EST databases, and identified more than 100 chemokine genes. These genes were CXC, CC and XC subfamily members, while no CX3C gene was identified. We also searched chemokine genes in pufferfish fugu and Tetraodon, and found only 18 chemokine genes in each species. The majority of the identified chemokine genes are unique to zebrafish or teleost fishes. However, several groups of chemokines are moderately similar to human chemokines, and some chemokines are orthologous to human homeostatic chemokines CXCL12 and CXCL14. Zebrafish also possesses a novel species-specific subfamily consisting of five members, which we term the CX subfamily. The CX chemokines lack one of the two N-terminus conserved cysteine residues but retain the third and the fourth ones. (Note that the XC subfamily only retains the second and fourth of the signature cysteines residues.) Phylogenetic analysis and genome organization of the chemokine genes showed that successive tandem duplication events generated the CX genes from the CC subfamily. Recombinant CXL-chr24a, one of the CX subfamily members on chromosome 24, showed marked chemotactic activity for carp leukocytes. The mRNA was expressed mainly during a certain period of the embryogenesis, suggesting its role in the zebrafish development. Conclusion: The phylogenic and genomic organization analyses suggest that a substantial number of chemokine genes in zebrafish were generated by zebrafish-specific tandem duplication events. During such duplications, a novel chemokine subfamily termed CX was generated in zebrafish. Only two human chemokines CXCL12 and CXCL14 have the orthologous chemokines in zebrafish. The diversification observed in the numbers and sequences of chemokines in the fish may reflect the adaptation of the individual species to their respective biological environment..
18. Nonaka S, Somamoto T, Kato-Unoki Y, Ototake M, Nakanishi T, Nakao M, Molecular cloning of CD4 from ginbuna crucian carp Carassius auratus langsdorfii, Fisheries Science, 10.1111/j.1444-2906.2008.01530.x, 74, 2, 341-346, 2008.04, The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii, a naturally occurring gynogenetic fish, is a useful model for studying T-cell-mediated immunity. CD4, a T-cell receptor (TCR) coreceptor, is a membrane-bound glycoprotein found on helper T-cells, and assists in the binding of major histocompatibility complexes. In the present study, full-length cDNAs encoding the CD4 molecule from the S3n strain of ginbuna crucian carp were cloned and characterized. 5′-rapid amplification of cDNA ends (RACE) and 3′-RACE yielded two distinct cDNA clones of CD4 homolog from the ginbuna, and these sequences share 95% identity at the amino acid level. These ginbuna CD4 molecules consisted of a signal peptide, immunoglobulin superfamily (IgSf) like domains, a transmembrane domain, and a cytoplasmic domain similar to other known CD4. A tyrosine protein kinase p56lck binding motif is conserved in the cytoplasmic tail of ginbuna CD4. Phylogenetic tree analysis indicated that ginbuna CD4 sequences are closely related to CD4L-1 from other fish species. Expression of ginbuna CD4 mRNA was detected in the gill, thymus, head kidney, trunk kidney and peripheral blood leukocytes, indicating that its expression pattern is similar to that of ginbuna TCRβ mRNA. The results suggest that ginbuna CD4 sequences are useful as molecular probes for helper T-cells..
19. Shin DH, Webb B, Nakao M, Smith SL, Molecular cloning, structural analysis and expression of complement component Bf/C2 genes in the nurse shark, Ginglymostoma cirratum, Dev Comp Immunol, 10.1016/j.dci.2007.03.001, 31, 11, 1168-1182, 2007.04, Factor B and C2 are serine proteases that provide the catalytic subunits of C3 and C5 convertases of the alternative (AP) and classical (CP) complement pathways. Two Bf/C2 cDNAs, GcBf/C2-1 and -2 (previously referred to as nsBf/C2-A and nsBf/C2-B), were isolated from the nurse shark, Ginglymostoma cirratum. GcBf/C2-1 and -2 are 3364 and 3082 bp in length and encode a leader peptide, three CCPs, one VWFA, the serine protease domain and have a putative factor D/C1s/MASP cleavage site. Southern blots show that there might be up to two Bf/C2-like genes for each of the two GcBf/C2 isoforms. GcBf/C2-1 and -2 are constitutively expressed, albeit at different levels, in all nine tissues examined. Expression in erythrocytes is a novel finding. Structural analysis has revealed that the localization of glycosylation sites in the SP domain of both putative proteins indicates that the molecular organization of the shark molecules is more like C2 than factor B. Phylogenetic analysis indicates that GcBf/C2-1 and -2 and TrscBf of Triakis scyllia (another shark species) originated from a common ancestor and share a remote ancestor with Bf and C2 of mammals and bony fish..
20. Nakao M, Kajiya T, Sato Y, Somamoto T, Kato-Unoki Y, Matsushita M, Nakata M, Fujita T, Yano T, Lectin pathway of bony fish complement: Identification of two homologues of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio), Journal of Immunology, 10.4049/jimmunol.177.8.5471, 177, 8, 5471-5479, 2006.10, The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway..
21. Somamoto T, Yoshiura Y, Sato A, Nakao M, Nakanishi T, Okamoto N, Ototake M., Expression profiles of TCRbeta and CD8alpha mRNA correlate with virus-specific cell-mediated cytotoxic activity in ginbuna crucian carp., Virology, 10.1016/j.virol.2006.01.019, 348, 2, 370-377, 2006.05, Our previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8α and TCRβ genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08-1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8α and TCRβ mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8α and TCRβ mRNAs. In addition, mRNA expressions of CD8α and TCRβ in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals..
22. Mutsuro J, Tanaka N, Kato Y, Dodds AW, Yano T, Nakao M, Two divergent isotypes of the fourth complement component from a bony fish, the common carp (Cyprinus carpio)., Journal of Immunology, 175, 7, 4508-4517, 175(7): 4508-4517, 2005.10, Duplication and diversification of several complement components is a striking feature of bony fish complement systems. It gives an interesting insight into an evolutionary strategy for the possible enhancement of the repertoire of innate immunity. The present study is aimed at examining diversity in bony fish C4, a member of the thioester-containing complement components. Two diverged cDNA sequences sharing only ∼32% identity at the amino acid level were isolated from the common carp and designated C4-1 and C4-2. C4-1 and C4-2 share a number of C4-like structural signatures, such as the thioester site and a disulfide-linked three-chain structure. Interestingly, they differ at the residue corresponding to the thioester-catalytic histidine, as seen in the human C4A and C4B isotypes, suggesting their distinct substrate specificities in the binding reaction of the thioester. Phylogenetic analysis indicates that the divergence of C4-1 and C4-2 predated the separation of the cartilaginous and bony fish lineages. Genomic Southern hybridization suggests the presence of single copy genes each encoding C4-1 and C4-2 in the carp genome. An activation fragment, C4a, was shown to be released from each isotype in carp serum activated via the classical aad/or lectin pathways. Synthetic peptides representing a putative C2 binding site on C4-1 and C4-2 inhibited the classical pathway-mediated hemolytic activity of carp seram in a dose-dependent manner. The results suggest that C4-1 and C4-2 represent two major lineages of C4 that are present in carp serum, have distinct binding specificities, and are functional in the classical/lectin pathways of complement activation..
23. Ishikawa J, Imai E, Moritomo T, Nakao M, Yano T, Tomana M, Characterisation of a fourth immunoglobulin light chain isotype in the common carp., Fish & Shellfish Immunology, 10.1016/j.fsi.2003.06.002, 16, 3, 369-379, 16 (3), 369-379, 2004.03, Three isotypes of immunoglobulin (Ig) light (L) chain, designated L1A, L1B, and L3, have been characterised in the common carp (Cyprinus carpio L.) to date. In this paper the molecular cloning of a fourth IgL isotype in carp, designated L2, is described. A reverse transcription-polymerase chain reaction (RT-PCR) method including 5′- and 3′-RACE was used to isolate carp L2 cDNA clones. The VL sequences could be divided into two distinct VL families, designated VL2-1 and VL2-2, most similar to rainbow trout (68% similarity) and zebrafish (78%) VL2 amino acid sequences, respectively. The CL amino acid sequences showed the highest similarity to zebrafish L2 (80%), and contained the characteristic cysteines necessary for intradomain or interchain disulphide bridges as did the VL sequences. Neither the VL nor CL sequences demonstrated such a high similarity to the other carp IgL isotypes, L1A, L1B, and L3. For JL segments, sequence variations appeared to be confined to a few positions. In the course of 5′- and 3′-RACE, cDNA clones containing recombination signal sequence (RSS), representatives of IgL sterile transcripts, were obtained. Southern blot analyses suggested that the locus of carp L2 has a cluster-like organisation. Phylogenetic analyses showed that both carp VL2 and CL2 amino acid sequences highly clustered with other teleost L2 sequences..
24. Nakao M, Uemura T, Yano T, Characterization of the soluble membrane attack complex (SMAC) of carp (Cyprinus carpio) complement., Journal of Faculty of Agriculture, Kyushu University, 48, 1-2, 127-134, 48: 127-134., 2003.01.
25. Nakao M, Fujiki K, Kondo M, Yano T, Detection of complement receptors on head kidney phagocytes of the common carp (Cyprinus carpio)., Fisheries Science, 10.1046/j.1444-2906.2003.00709.x, 69, 5, 929-935, 69: 927-933., 2003.01, Opsonization is a crucial function of the complement system in immune clearance. To identify receptors responsible for the complement-mediated opsonization in teleost, head kidney leukocytes from β-1,3-glucan-injected and non-injected common carp (Cyprinus carpio) were subjected to a rosette-forming assay with fixed sheep erythrocytes (Ef). Leukocyte fractions rich in granulocytes and macrophages from the glucan-injected carp had an ability to form the rosette with complement-coated Ef ut the corresponding fractions from non-injected fish did not, suggesting that β-1,3-glucan elicits expression of complement receptors on those cells. While sensitization of Ef with antibodies slightly elevated the rosette formation, complement-coated Ef showed a significant increase in rosette formation and this formation was inhibited with anti-carp C3. Rosette formation was also inhibited by EDTA but not by EGTA. These results suggest that complement-dependent opsonization in carp is mediated by CS-receptors analogous to mammalian complement receptor type 3 (CD11b/CD18 or integrin αM/β2), which recognizes the iC3b fragment in a divalent cation-dependent manner..
26. Fujiki K, Nakao M, Dixon B, Molecular cloning and characterization of a carp (Cyprinus carpio) cytokine-like cDNA that shares sequence similarity with IL-6 subfamily cytokines CNTF, OSM and LIF., Developmental and Comparative Immunology, 10.1016/S0145-305X(02)00074-5, 27, 2, 127-136, 27: 127-136., 2003.01, In the course of suppression subtractive hybridisation between sodium alginate-induced peritoneal cells (SA-PC) and normal head kidney cDNAs in common carp (Cyprinus carpio), a cytokine-like cDNA clone was found. The clone, named M17, contains a 1600bp nucleotide sequence that encodes a 215 amino acid putative protein that would have a pI of 9.01 and would include a 33 amino acid signal peptide. The 3′ untranslated region has seven ATTTA mRNA destabilising motifs that are common in cytokines and oncogenes. In a BLASTP search, M17 was most similar to chicken ciliary neurotrophic factor (CNTF) with 25% amino acid identity, followed by mammalian CNTF, cardiotrophin-1 and leukemia inhibitory factor (LIF) all of which belong to the IL-6 subfamily. However, M17 has some differences with CNTF in that CNTF has no signal sequence, the gene organisation of M17 is three exons and two introns, whereas that of CNTF is two exons and one intron, M17 has seven cysteines while CNTF has one cysteine, and M17 mRNA is detected in peripheral blood leukocytes as well as brain, whereas CNTF is expressed only in the nervous system. Compared to other members in the IL-6 subfamily cytokines, M17's cysteine positions and gene organisation are similar to those of oncostatin M and LIF, although amino acid identities are only 15-17%. Southern hybridisation suggested that M17 is a single copy gene. SA-PC showed significantly higher M17 mRNA levels than normal head kidney cells, which are considered to be a source of the SA-PC, indicating that M17 is inducible by inflammatory stimulation..
27. Nakao M, Hisamatsu S, Nakahara M, Kato Y, Smith SL, Yano T, Molecular cloning of the complement regulatory factor I isotypes from the common carp (Cyprinus carpio)., Immunogenetics, 10.1007/s00251-002-0518-9, 54, 11, 801-806, 54: 801-806., 2003.01, Factor I is a novel serine protease that regulates complement activation. Here we report the complete primary structure of two isotypic factor Is isolated from the common carp (Cyprinus carpio), a pseudotetraploid teleost. A carp hepatopancreas cDNA library was screened using two RT-PCR-amplified cDNA fragments encoding part of the carp factor I-like serine protease domain. Two distinct cDNA clones, designated FI-A and FI-B, were isolated. Their deduced amino acid sequences share 75.2% identity with each other. FI-A has a typical factor I-like domain organization composed of two disulfide-linked polypeptides (H-chain and L-chain). On the other hand, FI-B contains a novel sequence of 115 amino acids inserted at the N-terminus of the H-chain. Genomic Southern hybridization suggests that FI-A and FI-B are encoded by distinct genes in the carp genome. Expression analysis by RT-PCR revealed that the major site of FI-A expression is the ovary, whereas FI-B expression is detected mainly in the hepatopancreas at a level higher than that of FI-A. The present data, taken together, suggest that carp have duplicated genes coding for factor I, and FI-B with the novel insertion plays a dominant role in the complement system. In addition, homology search of the fugu genome database using the carp FI-A and FI-B sequences identified a putative fugu factor I gene, which has an exon/intron organization different from that of the human orthologue..
28. Kato Y, Nakao M, Mutsuro J, Zarkadis IK, Yano T (2003), The complement component C5 of the common carp (Cyprinus carpio) : cDNA cloning of two distinct isotypes that differ in a functional site., Immunogenetics, 10.1007/s00251-002-0528-7, 54, 11, 807-815, 54: 807-815., 2003.01, The complement component C5 plays important roles in inflammatory responses and complement-mediated cytolysis. In bony fish, although C5 has been identified at the DNA or the protein level in trout, carp and gilthead seabream, only partial C5 sequences are available. The present study was designed to obtain the complete primary structure of C5 from the common carp (Cyprinus carpio) and to examine its possible structural diversity. Reverse-transcribed polymerase chain reaction amplification from carp hepatopancreatic RNA resulted in isolation of six distinct C5-like cDNA segments, which were grouped into two divergent types (type I and type II). Using two sequences representative of the two types as probes, two distinct full-length cDNA clones (C5-1 and C5-2) were isolated, in addition to a truncated isoform of C5-1 (C5-1'). The deduced amino acid sequences of C5-1 and C5-2 share 83% identity and predict a typical two-chain structure of the mature protein that lacks the thioester bond, as in C5 from other animals. Southern hybridization of genomic DNA suggested the presence of multiple genes encoding C5-type I and a single gene encoding C5-type II. Interestingly, carp C5-type I contains novel subtypes like C5-1 that have a histidine instead of the well-conserved arginine at the cleavage site for the C5 convertase, both in the complete and truncated forms. Northern blotting analysis suggested that C5-type I and C5-type II are mainly expressed in hepatopancreas, and the expression levels are significantly increased by stimulating carp with lipopolysaccharide or β-1,3-glucan. Possible functional divergence among the C5 isotypes in carp is discussed..
29. Tomana M, Ishikawa J, Imai E, Moritomo T, Nakao M, Yano T, Characterization of immunoglobulin light chain isotypes in the common carp., Immunogenetics, 10.1007/s00251-002-0447-7, 54, 2, 120-129, 54: 120-129., 2002.01.
30. Nakao M, Matsumoto M, Nakazawa M, Fujiki K and Yano T, Diversity of complement factor B/C2 in the common carp (Cyprinus carpio): Three isotypes of B/C2-A expressed in different tissues., Developmental and Comparative Immunology, 10.1016/S0145-305X(01)00083-0, 26, 6, 533-541, 26: 533-541., 2002.01.
31. Shimasaki Y, Oshima Y, Yokota Y, Kitano T, Nakao M, Kawabata S, Imada N and Honjo T, Purification and identification of a tributyltin-binding protein from serum of Japanese flounder, Paralichthys olivaceus., Environmental Toxicology and Chemistry, 10.1897/1551-5028(2002)021<1229:PAIOAT>2.0.CO;2, 21, 6, 1229-1235, 21: 1229-1235., 2002.01.
32. Kono T, Fujiki K, Nakao M, Yano T, Endo M, Sakai M, The immune responses of common carp, Cyprinus carpio L., injected with carp interleukin-1b gene., Journal of Interferon and Cytokine Research, 10.1089/10799900252952190, 22, 4, 413-419, 22: 413-419., 2002.01.
33. Bayne CJ, Gerwick L., Fujiki K., Nakao M, Yano T, Immune relevant genes identified in the liver of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization., Developmental and Comparative Immunology, 10.1016/S0145-305X(00)00057-4, 25, 3, 205-217, 24: 205-217., 2001.01.
34. Fujiki K, Bayne CJ, Shin DH, Nakao M, Yano T, Molecular cloning of carp (Cyprinus carpio) C-type lectin and pentraxin by use of suppression subtractive hybridisation., Fish & Shellfish Immunology, 10.1006/fsim.2000.0331, 11, 3, 275-279, 11:275-279., 2001.01.
35. Nakao M, Osaka K, Kato Y, Fujiki K, Yano T, Molecular cloning of the complement C1r/C1s/MASP2-like serine proteases from the common carp (Cyprinus carpio)., Immunogenetics, 52, 3-4, 255-263, 52: 255-263., 2001.01.
36. Muturo J, Nakao M, Fujiki K, Yano T, Multiple forms of a2-macroglobulin from a bony fish, the common carp (Cyprinus carpio): Striking sequence diversity in functional sites., Immunogenetics, 10.1007/s002510000216, 51, 10, 847-855, 51: 847-855., 2000.01.
Presentations
1. Miki Nakao, Mucosal innate defense in carp fish involving T cells, complement system, and epithelial cells, The 2nd Asian Society of Developmental and Comparative Immunology Congress, 2022.10, Mucosal immunity is regarded as a crucial first-line defense against microbial infection in teleost fish, as supported by identification of well-developed mucosa-associated lymphoid organs, secretary forms of IgM and IgT, mucus lectins and antimicrobial peptides. Innate immune effector mechanisms, however, remain to be elucidated by functional evidence. We have identified CD8+ T cells of ginbuna crucian carp as effector cells to kill extracellular parasites, Ichthyophthirius multifiliis, in a contact-dependent and MHC-independent manner via serine proteases and perforin, suggesting the ancestral innate function of teleost T cells. On the other hand, presence of components of the complement system, a major humoral innate immune factor, in teleost skin has been reported with less information on their local activation and effector functions. Recently we have detected activation of the classical complement pathway towards Aeromonas hydrophila and A. salmonicida in carp skin mucus. This activation proceeds at least until the reaction step of C3, suggesting that the mucus complement is functional as an opsonic system. Interestingly, epithelial cells express a CD46-like regulator of complement activation (Tecrem) and stimulation of Tecrem seems to enhance robustness of epithelial sheet by up-regulating tight junction proteins in carp and ginbuna crucian carp. Since C3b, an activated form of C3, is a major ligand of Tecrem, mucosal complement activation may also contribute to strengthen the epithelial barrier against microbial infection. .
2. Rosli N, Yamamoto A, Nagasawa T, Somamoto T, Nakao M, Functional analysis of two complement C4 isotypes of carp using recombinant proteins, The 1st Congress of Asican Society of Developmental and Comparative Immunology, 2019.11, [URL].
3. Rapid purification of carp complement component C5 utilizing a specific immunoadsorbent.
4. Praparation of anti-zebrafish C5a to elucidate roles of complement C5a in inflammatory reaction of fish.
5. Nagasawa T, Somamoto T, Nakao M, Type I IFN production by carp thrombocytes as professional antiviral leukocytes, like mammalian plasmacytoid dendritic cells, via IRF signaling pathway, Asian Invertebrate Immunity Symposium, 2018.09.
6. Nakao M, Iwanaga S, Nagasawa T, Somamoto T, Phylogenetic inference on functions of the classical complement pathway in bony fish, 14th Congress of International Society of Developmental and Comparative Immunology, 2018.06, Bony fish is one of the ancestral vertebrates that possess immunoglobulins, on which the classical pathway of complement system relies for target recognition. To access the role of the classical pathway in bony fish complement, we have analyzed the hemolytic reaction and C3b-deposition on the target surface of the common carp (Cyprinus carpio) complement, using carp serum immunochemically depleted of factor D (Df), a serine protease responsible for activation of the alternative pathway, an activation and amplification cascade that bypasses the classical pathway. To our surprise, the Df-depletion abolished hemolytic activities of the serum both through the classical and alternative pathways, when assayed using antibody-sensitized and non-sensitized sheep and rabbit erythrocytes. However, a low level of C3b-deposition, probably resulted from the classical pathway activation in the absence of Df was demonstrated on various target surfaces by flow cytometry and ELISA. In addition, Df-depletion inhibited C5-deposition essential for the cytotoxic complex formation on the target cells. The C3b-bound erythrocytes were hemolyzed by serum but easily lysed by Mg-EGTA-serum, probably triggering the alternative pathway amplification more efficiently than unbound erythrocytes. These results, taken together, suggest that an ancestral classical pathway alone lacks C5-activating ability but tags target cells with a small amount of C3b, which triggers enhanced C3-activation by the alternative pathway, leading to completion of the terminal cytolytic pathway..
7. Nagasawa T, Somamoto T, Nakao M, Viral ligands recognition of carp thrombocytes as natural type I interferon producing cells, 14th Congress of International Society of Developmental and Comparative Immunology, 2018.06.
8. Cytotoxicity of T cells against a prasite Ichthyophthirius multiliis in ginbuna crucian carp.
9. Cytotoxicity of T cells against a prasite Ichthyophthirius multiliis in ginbuna crucian carp.
10. Nakao M, Noguchi M, Akahoshi S, Nagasawa T, Somamoto T, Functional diversity of two C7 isotypes in bony fish, a primitive vertebrate model, European Meeting on Complement in Human Disease, 2017.09, [URL].
11. Prakash H, Motobe S, Nagasawa T, Somamoto T, Nakao M, Functional analysis of Tecrem, a CD46-like complement regulatory protein, on epithelial cells in the common carp, The JSFS 85th Anniversary-Commemorative International Symposium, 2017.09.
12. Development of KHV disease in ginbuna crucian carp by suppression of cellular immunity..
13. Cytotoxicity of T cells against a prasite Ichthyophthirius multiliis in ginbuna crucian carp.
14. Functional analysis of two C7 isotypes of carp complement system..
15. Harsha Prakash, Shiori Motobe, Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao, Expression and homeostatic functions of Tecrem, a CD46-like complement regulatory protein on epithelial cells in bony fish., 26th International Complement Workshop, 2016.09, Mammalian CD46 has been reported as a multitasking immune modulator, which regulates complement activation on host cells, T cell-mediated adaptive responses, and wound repair by involving epithelial cells. It is of particular interest to explore the evolutionary path of such versatile functions of CD46.

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. Furthermore, we have also found that Tecrem expressed on T cells modulates mitogen-induced T cell proliferation in ginbuna crucian carp, indicating that modulation of adaptive immunity is one of the evolutionarily conserved functions of the CD46-like complement regulator. 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 ELISA-based assay for Tecrem 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.

Towards further functional analyses of carp Tecrem, we have prepared recombinant Tecrem proteins in the bacterial expression system. 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. Interaction of the two recombinant domains with carp C3 isotypes and their inhibition of carp complement activation cascades are currently analyzed in parallel with raising polyclonal antibodies, for the clarification of functional modules and their mode of action.
.
16. Kazuki Yoshioka, Yoko Kato-Unoki, Takahiro Nagasawa, Tomonori Somamoto, Miki Nakao, Carp properdin: structural and functional diversity of two isotypes., 26th International Complement Workshop, 2016.09, The alternative pathway (ACP) of the complement system is an antibody-independent activation pathway, in which properdin (Pf) has been known as a positive regulator of the activation and a possible pattern-recognition molecule to trigger ACP activation. Teleost complement system has a striking feature that some of its components are diversified into multiple isoforms with different functions. However this diversity is less characterized for teleost Pf, especially at the protein level. The present study was aimed at elucidating isotypic diversity and functional differentiation of Pf in the common carp. Molecular cloning of carp Pf revealed two distinct full-length cDNA sequences, CaPf1 and CaPf2, that predicts mature proteins composed of seven thrombospondin type1 domains (TSP0-TSP6), sharing 77% amino acid sequence identity. Genomic Southern hybridization suggested that CaPf1 and CaPf2 are encoded by single each gene in carp genome. Real-time quantitative PCR indicated that expression level of CaPf1 is most abundant in the spleen, whereas CaPf2 was detected mainly in head kidney and renal kidney. Rabbit antibodies were raised against their recombinant proteins corresponding to TSP4-6 domains. Western blotting using anti-CaPf1 and anti-CaPf2 revealed that CaPf1 and CaPf2 are mainly present as a hexamer of polypeptide with molecular weights of 49,000 and 48,000, respectively, in carp serum. Interestingly, CaPf1 and CaPf2 showed different spectra of binding to various microbes, suggesting their functional diversity..
17. Gene knockout of myeloperoxidase in zebrafish that lacks adaptive immunity.
18. Effects of dietary soybean meal protein on antibody response in fish.
19. Kazuki Yoshioka, Yoko Kato-Unoki, Tomonori Somamoto, Miki Nakao, Structural and functional diversity of properdin isotypes in the common carp complement system. , 13th Congress of the International Society for Developmental and Comparative Immunology, 2015.06, The alternative pathway (ACP) of the complement system is an antibody-independent activation pathway, in which properdin (Pf) has been known as an essential positive regulator of the activation and possibly as a pattern-recognition molecule to trigger ACP activation. Teleost complement system has a striking feature that some of its components are diversified into multiple isoforms with different functions. However this diversity is less characterized for teleost Pf, especially at the protein level. The present study was aimed at elucidating isotypic diversity and functional differentiation of Pf in the common carp. Molecular cloning of carp Pf revealed two distinct full-length cDNA sequences, CaPf1 and CaPf2, that predicts mature proteins composed of seven thrombospondin type1 domains (TSP0-TSP6), sharing 77% amino acid sequence identity. Genomic Southern hybridization suggested that CaPf1 and CaPf2 are encoded by single each gene in carp genome. Real-time quantitative PCR indicated that expression level of CaPf1 is most abundant in the spleen, whereas CaPf2 was detected mainly in head kidney and renal kidney. Rabbit antibodies were raised against their recombinant proteins corresponding to TSP4-6 domains. Western blotting using anti-CaPf1 and anti-CaPf2 revealed that CaPf1 and CaPf2 are mainly present as a hexamer of polypeptide with molecular weights of 49,000 and 48,000, respectively, in carp serum. Interestingly, CaPf1 and CaPf2 showed different spectra of binding to various microbes, suggesting their functional diversity..
20. Immune-regulatory mechanism in rag1-deficient zebrafish.
21. Effect of soy bean meal feeding on the complement system of rainbow trout.
22. Indriyani Nur, 原田光利, 杣本 智軌, 中尾 実樹, 中村亮太, 辻倉正和, Characterizations of membrane-bound complement regulatory protein in Ginbuna Crucian Carp Carassius auratus langsdorfii, 日本比較免疫学会学術集会, 2013.08.
23. Carp thrombocytes are activated by other leukocytes to show phagocytosis..
24. Takahiro Nagasawa, Chihaya Nakayasu, Tomomasa Matsuyama, Aja M. Rieger, Daniel R. Barreda, Tomonori Somamoto, Miki Nakao, Phagocytosis and bactericidal abilities of teleost thrombocytes, 12th Congress of International Society of Developmental and Comparative Immunology, 2012.07, Thrombocytes have been recognized as haemostatic cells in non-mammalian vertebrates. Unlike mammalian
platelets, thrombocytes are nucleated cells with a lymphocyte-like morphological feature, and possible
involvement of thrombocytes in innate immune function has been considered in addition to the haemostatic
function. In the present study, we report phagocytic abilities of some teleost thrombocytes. Using a monoclonal
antibody specific for thrombocytes of common carp (Cyprinus carpio), thrombocytes were isolated from
peripheral blood and examined for expression of various immune-related genes, resulting in detection of
significant level of lysozyme, iNOS and MHC class II using RT-PCR. Upon flow cytometry-based phagocytosis
assay, a number of thrombocytes ingested fluorescent latex particles (0.5 μm, 1 μm, 2 μm and 3 μm), bacteria
(Escherichia coli) and zymosan particles. Phagocytosis by the thrombocytes was also confirmed by fluorescent
microscopy and transmission electron microscopy, which revealed internalization of these particles into
thrombocytes. We also observed that thrombocytes of the olive flounder (Paralichthys olivaceus) had similar
phagocytic behavior. These data indicate that thrombocytes of those species are potent phagocytes, suggesting
that those phagocytic characteristics of thrombocytes are widely conserved in teleosts. We also assessed a
phagolysosome formation ability of teleost thrombocytes against the ingested pathogens, for detecting
intracellular bactericidal activities of those thrombocytes. By using an ImageStream multi-spectral flow
cytometer, we assessed phagolysosome fusion of goldfish (Carassius auratus) thrombocytes. On this analysis
we detected that lysosomes of these thrombocytes visualized with fluorescent dextran were co-localized with the
ingested small beads and formed a ring around large beads like other typical phagocytes. Overall, the results
indicate that teleost thrombocytes have dual functions as not only haemostatic cells but also as phagocytic
immune cells against microbial infections..
25. Haruka Tsukamoto, Yutaka Fukuda, Tomonori Somamoto, Miki Nakao, Functions of CD8-positive and CD4-positive lymphocytes against virus-infection in ginbuna crucain carp, 12th Congress of International Society of Developmental and Comparative Immunology, 2012.07, Two serotypes of Streptococcus parauberis, pathogens of streptococcosis in Japanese flounder (Paralichthys
olivaceus), have caused severe losses of the livestock, due to the lack of effective vaccine and its resistance to
antibiotics approved for flounder culture. These strains show different virulence, but their mechanisms of their
pathogenicity are totally unknown, and identification of virulence factors are needed for effective vaccine
development. The present study, therefore, was aimed at clarifying effect of S. parauberis culture supernatant on
flounder. Since the diseased flounder shows anaemia, we examined the effects of S. parauberis on flounder
peripheral blood. As a result, S. parauberis -injected flounder showed decreased numbers of peripheral
erythrocytes, suggesting that S. parauberis may produce a haemolytic factor. To characterize the haemolytic
factor, a supernatant of S. parauberis culture (25°C for 48h in Todd Hewitt broth) was mixed with sheep
erythrocytes, resulting in significant level of haemolysis. The haemolytic factor was stable on heating at 100°C
for 10 min, and passed through an ultrafiltration membrane with a 5 kDa cut off limit (Amicon Ultra15),
indicating that the factor is not proteinaceous haemolysin but a heat-resistant low molecular mass substance. We
also investigated the effects of the culture supernatant on flounder leukocytes. Peripheral leukocytes were
separated from flounder and carp using Percoll discontinuous density gradient centrifugation and cultured with
the supernatant in 96-well plate. The stimulated flounder cells showed agglutination and significant proliferation
as assayed by BrdU uptake, while carp cells did not. These results suggest that S. parauberis secrets a mitogen
specific for flounder leucocytes. Characterization of the mitogen and resulting leucocyte response is in progress..
26. Tomokazu Yamaguchi, Kazufumi Takamune, Masakazu Kondo, Yukinori Takahashi, Miki Nakao, Yoko Kato-Unoki, Tamotsu Fujii, Identification of Pattern Recognition Molecules in Hagfish Complement System, 12th Congress of International Society of Developmental and Comparative Immunology, 2012.07, All extant jawed vertebrates share a common adaptive immune system in which immunoglobulin domain-based
molecules act as antigen receptors. On the other hand, jawless vertebrates, lamprey and hagfish, use variable
lymphocyte receptors composed of leucine-rich repeat cassettes for antigen recognition. Since invertebrates and
primitive chordates do not have an adaptive immune system, the immune system seems to change dramatically
in the course of evolution from primitive chordates to gnathostomes. From a phylogenic perspective of defence
mechanisms, previously we found complement C3 and mannose-binding lectin-associated serine protease 1
(MASP-1) in hagfish and suggested the involvement of lectin pathway in hagfish innate immune system. In this
study, we focused on the pattern recognition molecules in the hagfish, Eptatretus burgeri, and tried to purify
them from serum by affinity chromatography. When the serum was treated with GlcNAc-agarose, followed by
successive elution of the binding molecules with GlcNAc and EDTA, mainly four proteins (31 kDa, 27 kDa, 26
kDa, and 19 kDa) and 26 kDa protein were detected in the GlcNAc-eluate and EDTA-eluate, respectively.
Collagenase treatment showed the presence of collagen-like domain only in the 26 kDa protein in the EDTAeluate.
Since common pattern recognition molecules such as mannose-binding lectins possess collagen-like
domains, we examined the entity of the 26 kDa protein by sequencing its N-terminal amino acids and cDNA
obtained by 3’ and 5’ RACE methods, and identified it as a member of C1q family. Herein, it will be referred to
as hagfish C1q (hagC1q). Western blot analyses using anti-hagC1q, MASP-1, and complement C3 antibodies
showed that hagC1q associated with MASP-1 and complement C3 in the serum and had binding ability to
Escherichia coli as a divalent cation-dependent manner. These results suggest that hagC1q plays an important
role in hagfish innate immune system..
27. Vo Kha Tam, Chie Okura, Masakazu Kondo, Tomonori Somamoto, Miki Nakao, Isotypic diversity in the ontogenetic expression of the complement component in the common carp (Cyprinus carpio), 12th Congress of International Society of Developmental and Comparative Immunology, 2012.07, Isotypic diversity of complement components is a striking feature of the teleost complement system. As a first
line of innate defence, the complement system has been considered as an important clue to humoral defence in
early development however functional diversity of the isotypes during teleost ontogeny is poorly understood.
The present study aimed at clarifying comprehensive picture of ontogenetic expression of the diversified
complement component isotypes in carp. Real-time quantitative PCR detected embryonic expression of C1r/s,
MASPs, factor B/C2, C3, C4, C5, C6, C7, C8, C9, and factor I. A remarkable difference in the expression time
course was noted between the isotypes in C3, C4, and C5. Especially, teleost-specific isotypes of C3 and C4
(non-histidine-type) started around hatching, in contrast to evolutionarily common isotypes (histidine-type),
which showed much earlier expression. Whole-mount in situ hybridization of carp embryos revealed some
difference in embryonic expression sites of two major C3 isotypes (C3-H1 and C3-S) in addition to common
expression sites such as the yolk syncytial layer. The temporal and spatial differences in expression among the
isotypes suggest that the isotypes are functionally differentiated in teleost early development..
28. Miki Nakao, Masakazu Tsujikura, Satoko Ichiki, Vo Kha Tam, Tomonori Somamoto, Structural and functional diversity of the complement system, an innate immune factor, in fish, 8th International Congress of Comparative Physiology and Biochemistry, ICCPB-Nagoya, 2011.06.
29. Miki Nakao, Satoko Ichiki, Masakazu Tsujikura, Vo Kha Tam, Tomonori Somamoto, Complement system in teleost fish: Isotypic diversity in pathogen-recognition, activation cascade, and ontogeny, Comparative Immunology and Pathology Workshop Edmonton 2011, 2011.05.
30. Takahiro Nagasawa, Chihara Nakayasu, Tomomasa Matsuyama, Tomonori Somamoto, Miki Nakao, Phagocytic activities of carp (Cyprinus carpio ) thrombocytes, Comparative Immunology and Pathology Workshop Edmonton 2011, 2011.05.
31. Phagocytic function of fish thrombocytes.
32. Diversity in target-recognition of carp serum collectins related to the lectin pathway of the complement
Akito Ichiki, Haruka Tsukamoto, Tomonori Somamoto, and Miki Nakao.
33. Functional diversity of the complement component isotypes in bony fish innate immunity: a model study using the common carp.
Nakao, M., Ichiki, S., Mutsuro, J., Tsujikura, M., and Somamoto, T..
34. Functional differentiation of C7 isotypes in the common carp complement system.
35. Recognition specificity of carp serum lectins associated with the complement system.
36. Structural diversity and functional differentiation of the complement components in bony fish.
Miki Nakao.
37. Subcomponent structure of the first complement component (C1) in the common carp.
Takashi Ichii, Masakazu Tsujikura, Tomonori Somamoto, Yoko Kato-Unoki, Shin-ichi Kato, Michiyasu Yoshikuni, Miki Nakao.
38. Expression pattern of complement component C3 isotypes during early development of the common carp.
Vo Kha Tam, Chie Okura, Tomonori Somamoto, Miki Nakao.
39. Functional differentiation of complement C3 isotypes in the teleost immune defense.
Satoko Ichiki, Yoko Kato-Unoki, Tomonori Somamoto, Miki Nakao.
40. cDNA cloning and expression analysis of Fas ligand in ginbuna crucian carp.
41. Phagocytic ability of carpo thrombocytes.
42. Subcomponent structure of the first complement component (C1) in the common carp.
43. Cloning, purification and characterization of zymosan-binding proteins in Nile Tilapia (Oreochromis niloticus)
Soha Gomaa, Tomonori Somamoto and Miki Nakao.
44. Complement components constituting the classical activation pathway, an antibody-dependent activation cascade of the complement system..
45. cDNA cloning of four lineages of MHC class I genes in a cell line from clonal ginbuna crucian carp, and their expression in the cell line infected with CHNV.
46. Identification of a novel membrane-bound regulator of complement activation in carp and zebrafish.
47. Diversity of carp serum lectins involved in the lectin pathway of the complement activation..
48. Identification and expression analysis of two isotypes of carp complement component C7.
49. Structural and functional diversity of the complement components in bony fish: implication for innate immune defense.
50. Structure and regulation of the isotypic genes of the complement factor B in the common carp.
51. Identification of a novel complement regulatory factor in bony fish.
52. Structure and regulation of the isotypic genes of the complement factor B in the common carp.
53. Antibacterial substances from bony fish: structure, function, and application.
54. Functional analysis of carp C3 isotypes using monoclonal antibodies.
55. Identification and expression analysis of a novel interleukine 8 (IL-8)-like CXC chemokine in carp (Cyprinus carpio).
56. Expressed sequence tag analysis of kidney and spleen from ginbuna crucian carp,
Carassius auratus langsdorfii
.
57. cDNA cloning of MHC class I and MHC-related genes in a cell line from clonal ginbuna crucian carp.
58. Expression analysis of complement component isotypes during early development of the common carp.
59. Identification of Carp Complement Component C1.
60. Functional analysis of carp C3 isoforms using monoclonal antibodies.
61. Identification of novel regulators of complement activation in fish.
62. Recombinant carp complement component C4 and C5: Expression and functional analysis.
63. Identification and expression analysis of zebrafish complement component genes.
64. Inference of bio-defensive functions of the complement system based on its gene expression pattern..
65. EST Analysis of mRNAs Expressed in Gill and Intestine of Carp (Cyprinus carpio. L).
66. Identification of the genes encoding complement components from the zebrafish genome database.
67. Molecular cloning of C3d, a fragment from complement component C3 with possible adjuvant activity, from marine cultured fish.
68. Molecular cloning of a complement-regulatory factor from zebrafish.
69. Molecular cloning of a novel complement-regulatory factor from the common carp.
70. Molecular cloning of C1q A, B and C chains from common carp.
71. DIVESIFICATION OF COMPLEMENT COMPONENT ISOTYPES IN THE COMMON CARP: EXPRESSION PATTERN AND SOME FUNCTIONAL ASPECTS.
72. MOLECULAR CLONING AND CHARACTERIZATION OF CD4 IN GINBUNA CRUCIAN CARP.
73. Molecular cloning and characterization of the complement C1q A, B, and C chains in common carp .
74. THE COMPLEMENT SYSTEM IN INVERTEBRATES AND LOWER VERTEBRATES.
Awards
  • Diversification and functional significance of the complement component isotypes in bony fish.
Educational
Educational Activities
Undergraduage course: Fundamental Chemistry B, Introduction to Animal Production Science, Fish Immunology, Marine Biochemistry, Biochemical Experiment training, Marine Biochemical Experiment training.

Master course: Molecular and Cellular Biochemistry, Comparative Immunology
Social
Professional and Outreach Activities
Simple and rapid methods to monitor the degree of fish health are required to produce cultured fish. I have studied if serum complement acitivity is a good measure to estimate the level of bio-defense activity of cultured fish, aiming at development of a convenient kit for fish complement assay. Several natural materials have been tested for their possible immunostimulation..