- Email: [email protected]
Chronic saline exposures interfere with basal and induced immunity in striped catfish (Pangasianodon hypophthalmus, sauvage) exposed to virulent strain of Edwardsiella ictaluri
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
E-mail address: [email protected] (A.M. Ferreira). * These authors have contributed equally to this work.
O-090. Immune responses to alum in the Japanese pufferfish (Takifugu rubripes) Masahiro Sakai 1, Takashi Morimoto 1, Gouranga Biswas 2, Tomoya Kono 1, Jun-ichi Hikima 1, * 1 Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan 2 Kakdwip Research Centre of ICAR-Central Institute of Brackishwater Aquaculture, Kakdwip, West Bengal 743347, India
Abstract To elucidate the basis of innate immune responses against insoluble compounds (such as silica and alum) through the inflammasome activation pathway in fish, biological assays and expression analysis of immunerelated genes were conducted in the Japanese pufferfish (Takifugu rubripes) head kidney (HK) cells. At first, the Japanese pufferfish was stimulated by intraperitoneal injection of alum (4 mg/fish). After 0, 6, 12, 24 and 48 h post-injection, HK cells were prepared from the injected fish. To understand functionality of activated inflammatory response, phagocytic activity and superoxide anion production (NBT assay) in the HK cells were assessed. As results, the phagocytic and NBT reduction activities were significantly increased in alum-stimulation. To further clarify the immune response through transcriptomic analysis, expression of 19 cytokine genes was checked by the multiplex reverse transcription (RT)-PCR method. Results showed that expression of IL-6 and type I-IFN genes was significantly elevated in the alum-stimulated fish. Furthermore, expression of NLR-C9 and NLR-C12 genes [i.e., NOD-like receptor (NLR) family genes as sensor molecules of inflammasome], ASC and caspase-1 genes (inflammasome associated molecules) were examined by qPCR. Results indicated an increased expression of four different inflammasome associated genes in the HK cells of fish injected with alum. The results suggested activation of inflammatory response to the alum with a likely involvement of inflammasome components in the Japanese pufferfish. Keywords: Inflammasome, NLR family genes, cytokine, gene expression, alum, Japanese pufferfish * Corresponding author. Tel/Fax: þ81 985 58 7230. E-mail address: [email protected] (J.-ichi Hikima).
O-091. Mannose-specific immunoglobulin M in the mucus of fugu Takifugu rubripes is utilized by a monogenean parasite Heterobothrium okamotoi for host recognition Shigeyuki Tsutsui 1, Kento Igarashi 1, Sachi Hirakawa 2, Sho Hosoya 3, Hiroaki Suetake 2, Kiyoshi Kikuchi 3, Yuzuru Suzuki 3, Osamu Nakamura 1, Satoshi Tasumi 3, Toshiaki Miyadai 2 1
School of Marine Biosciences, Kitasato University, Japan 2 Faculty of Marine Bioscience, Fukui Prefectural University, Japan 3 Fisheries Laboratory, University of Tokyo, Japan Abstract How parasites recognize their definitive hosts is a mystery; however, it is likely that parasitism is initiated through the molecular recognition of certain molecules on host surfaces by parasites. Fish ectoparasites make the initial contact with their hosts at the body surface, including the skin and gills, which is covered with a mucous layer. Fish body mucus contains several types of molecules acting as defense factors. The present study provides new evidence demonstrating that mannose-specific IgM in the gill mucus is utilized by a monogenean parasite, Heterobothrium okamotoi, to recognize their host, fugu (Takifugu rubripes). First, we determined that
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the infective parasite larvae, the oncomiracidium, shed their cilia and metamorphosed into juveniles when exposed to mannose-binding proteins partially purified from fugu skin or skin mucus. These proteins were identified as mannose-specific IgM and two mannose-binding lectins, pufflectin and kalliklectin. Among these, deciliation was significantly induced by IgM and inhibited by mannose or the specific antibody against fugu IgM, whereas the other proteins had no effect. Immunofluorescent staining revealed that fugu mannose-specific IgM bound to the ciliated epithelial cells of the oncomiracidium. In contrast, IgM without mannose affinity induced deciliation at significantly lower levels. These findings suggest that the binding of fugu IgM to a ligand present on the cell surface via the antigen-binding site triggered deciliation of oncomiracidium. The mannose-binding IgM concentration in the gill mucus where the parasite colonized was high enough to induce deciliation in vitro, indicating that H. okamotoi initially uses the defense molecule for establishing parasitism. Keywords: Fugu, Heterobothrium okamotoi, Host recognition, IgM, Mannose, Parasite.
O-092. Chronic saline exposures interfere with basal and induced immunity in striped catfish (Pangasianodon hypophthalmus, sauvage) exposed to virulent strain of Edwardsiella ictaluri M elodie Schmitz, Syaghalirwa NM. Mandiki, Jessica Douxfils, Tamar Ziv, Arie Admon, Patrick Kestemont de Chimie Physique The orique et Structurale, University of Unite Namur (UNamur), Rue de Bruxelles 61, 5000 Namur, Belgium Abstract Hyperosmotic stress has often been investigated from osmoregulation perspectives while the effects of such stress on the immune capacity remain largely unexplored. In this study, stenohaline striped catfish Pangasianodon hypophthalmus were submitted to gradual hyperosmotic stress during 20 days up to 10 and 20 ppt. Hyperosmotic stress was followed by an infection with a highly virulent strain of an intracellular gram negative bacteria, Edwardsiella ictaluri, responsible for Enteric Septicemia of Catfish (ESC). Osmoregulatory (i.e. plasma osmolarity and NaþKþ ATPase activity), immune (i.e. lysozyme, complement, respiratory burst, white blood cells, heat shock protein and high-mobility group B1 protein) parameters and mortality rates were monitored. Moreover, a label free quantitative proteomics workflow was performed to study how salinity affects the proteome of kidney in healthy and infected catfish. The flow consisted in initial global profiling of relative peptide abundances (by LC/MS, peak area quantification based on extracted ion currents) followed by MS/MS identification. In total, 496 differentially expressed proteins were analyzed in DAVID interface to isolate functional pathways and categories involved in the response to bacterial infection and which were influenced by salinity changes. Pathways and functional categories, particularly those related to stress, immunity and ion homeostasis, are discussed. We hypothesized that prolonged exposure to mild and high hyperosmotic stress may induce chronic sterile inflammation and disruption of immune homeostasis that may lead to excessive inflammatory response and serious ESC disease. Keywords: Pangasianodon hypophalmus, salinity, infectious disease, immunity, osmoregulation, proteomics.
O-093. Morphological characterization via light and electron microscopy of atlantic jackknife clam (Ensis directus) hemocytes Brian M. Preziosi§, Timothy J. Bowden School of Food and Agriculture, Aquaculture Research Institute, University of Maine, Hitchner Hall, Orono, ME 04469, USA Abstract
E-mail address: [email protected] (A.M. Ferreira). * These authors have contributed equally to this work.
O-090. Immune responses to alum in the Japanese pufferfish (Takifugu rubripes) Masahiro Sakai 1, Takashi Morimoto 1, Gouranga Biswas 2, Tomoya Kono 1, Jun-ichi Hikima 1, * 1 Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan 2 Kakdwip Research Centre of ICAR-Central Institute of Brackishwater Aquaculture, Kakdwip, West Bengal 743347, India
Abstract To elucidate the basis of innate immune responses against insoluble compounds (such as silica and alum) through the inflammasome activation pathway in fish, biological assays and expression analysis of immunerelated genes were conducted in the Japanese pufferfish (Takifugu rubripes) head kidney (HK) cells. At first, the Japanese pufferfish was stimulated by intraperitoneal injection of alum (4 mg/fish). After 0, 6, 12, 24 and 48 h post-injection, HK cells were prepared from the injected fish. To understand functionality of activated inflammatory response, phagocytic activity and superoxide anion production (NBT assay) in the HK cells were assessed. As results, the phagocytic and NBT reduction activities were significantly increased in alum-stimulation. To further clarify the immune response through transcriptomic analysis, expression of 19 cytokine genes was checked by the multiplex reverse transcription (RT)-PCR method. Results showed that expression of IL-6 and type I-IFN genes was significantly elevated in the alum-stimulated fish. Furthermore, expression of NLR-C9 and NLR-C12 genes [i.e., NOD-like receptor (NLR) family genes as sensor molecules of inflammasome], ASC and caspase-1 genes (inflammasome associated molecules) were examined by qPCR. Results indicated an increased expression of four different inflammasome associated genes in the HK cells of fish injected with alum. The results suggested activation of inflammatory response to the alum with a likely involvement of inflammasome components in the Japanese pufferfish. Keywords: Inflammasome, NLR family genes, cytokine, gene expression, alum, Japanese pufferfish * Corresponding author. Tel/Fax: þ81 985 58 7230. E-mail address: [email protected] (J.-ichi Hikima).
O-091. Mannose-specific immunoglobulin M in the mucus of fugu Takifugu rubripes is utilized by a monogenean parasite Heterobothrium okamotoi for host recognition Shigeyuki Tsutsui 1, Kento Igarashi 1, Sachi Hirakawa 2, Sho Hosoya 3, Hiroaki Suetake 2, Kiyoshi Kikuchi 3, Yuzuru Suzuki 3, Osamu Nakamura 1, Satoshi Tasumi 3, Toshiaki Miyadai 2 1
School of Marine Biosciences, Kitasato University, Japan 2 Faculty of Marine Bioscience, Fukui Prefectural University, Japan 3 Fisheries Laboratory, University of Tokyo, Japan Abstract How parasites recognize their definitive hosts is a mystery; however, it is likely that parasitism is initiated through the molecular recognition of certain molecules on host surfaces by parasites. Fish ectoparasites make the initial contact with their hosts at the body surface, including the skin and gills, which is covered with a mucous layer. Fish body mucus contains several types of molecules acting as defense factors. The present study provides new evidence demonstrating that mannose-specific IgM in the gill mucus is utilized by a monogenean parasite, Heterobothrium okamotoi, to recognize their host, fugu (Takifugu rubripes). First, we determined that
89
the infective parasite larvae, the oncomiracidium, shed their cilia and metamorphosed into juveniles when exposed to mannose-binding proteins partially purified from fugu skin or skin mucus. These proteins were identified as mannose-specific IgM and two mannose-binding lectins, pufflectin and kalliklectin. Among these, deciliation was significantly induced by IgM and inhibited by mannose or the specific antibody against fugu IgM, whereas the other proteins had no effect. Immunofluorescent staining revealed that fugu mannose-specific IgM bound to the ciliated epithelial cells of the oncomiracidium. In contrast, IgM without mannose affinity induced deciliation at significantly lower levels. These findings suggest that the binding of fugu IgM to a ligand present on the cell surface via the antigen-binding site triggered deciliation of oncomiracidium. The mannose-binding IgM concentration in the gill mucus where the parasite colonized was high enough to induce deciliation in vitro, indicating that H. okamotoi initially uses the defense molecule for establishing parasitism. Keywords: Fugu, Heterobothrium okamotoi, Host recognition, IgM, Mannose, Parasite.
O-092. Chronic saline exposures interfere with basal and induced immunity in striped catfish (Pangasianodon hypophthalmus, sauvage) exposed to virulent strain of Edwardsiella ictaluri M elodie Schmitz, Syaghalirwa NM. Mandiki, Jessica Douxfils, Tamar Ziv, Arie Admon, Patrick Kestemont de Chimie Physique The orique et Structurale, University of Unite Namur (UNamur), Rue de Bruxelles 61, 5000 Namur, Belgium Abstract Hyperosmotic stress has often been investigated from osmoregulation perspectives while the effects of such stress on the immune capacity remain largely unexplored. In this study, stenohaline striped catfish Pangasianodon hypophthalmus were submitted to gradual hyperosmotic stress during 20 days up to 10 and 20 ppt. Hyperosmotic stress was followed by an infection with a highly virulent strain of an intracellular gram negative bacteria, Edwardsiella ictaluri, responsible for Enteric Septicemia of Catfish (ESC). Osmoregulatory (i.e. plasma osmolarity and NaþKþ ATPase activity), immune (i.e. lysozyme, complement, respiratory burst, white blood cells, heat shock protein and high-mobility group B1 protein) parameters and mortality rates were monitored. Moreover, a label free quantitative proteomics workflow was performed to study how salinity affects the proteome of kidney in healthy and infected catfish. The flow consisted in initial global profiling of relative peptide abundances (by LC/MS, peak area quantification based on extracted ion currents) followed by MS/MS identification. In total, 496 differentially expressed proteins were analyzed in DAVID interface to isolate functional pathways and categories involved in the response to bacterial infection and which were influenced by salinity changes. Pathways and functional categories, particularly those related to stress, immunity and ion homeostasis, are discussed. We hypothesized that prolonged exposure to mild and high hyperosmotic stress may induce chronic sterile inflammation and disruption of immune homeostasis that may lead to excessive inflammatory response and serious ESC disease. Keywords: Pangasianodon hypophalmus, salinity, infectious disease, immunity, osmoregulation, proteomics.
O-093. Morphological characterization via light and electron microscopy of atlantic jackknife clam (Ensis directus) hemocytes Brian M. Preziosi§, Timothy J. Bowden School of Food and Agriculture, Aquaculture Research Institute, University of Maine, Hitchner Hall, Orono, ME 04469, USA Abstract