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NO43 - 1 (2008)

Review: Cellular Defense Mechanisms in Bivalve Molluscs

<>Keisuke G. Takahashi* and Kiyokuni Muroga<>Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan
(Received May 14, 2007)
ABSTRACT柚any bivalve species are cultured widely around the world due to their commercial importance. As a result of the increases in areas and production associated with intensive culture, mass mortalities of bivalves caused by a wide variety of agents have become a serious problem. Various infectious diseases represented by protozoan and bacterial diseases have been threatening shellfish culture industries, and hence, many investigations have been made on the defense mechanisms, as well as diseases, in bivalves. The molluscan defense mechanism is comprised of the only innate immunity, which is also dependent on cellular components such as hemocytes possessing phagocytic and bactericidal activities and on humoral defense molecules such as lectin and lysozyme. In this review, we summarize cellular defense mechanisms in bivalve molluscs focusing on hemocytes: 1) morphology and classification, 2) functional profiles (phagocytosis, encapsulation, wound repair), 3) reactive oxygen species, 4) effects of natural and anthropogenic environmental factors, and 5) stress and signaling pathways.

Key words: bivalve mollusc, cellular defense mechanism, hemocyte, phagocytosis, reactive oxygen species, ROS, environmental factor, stress reaction

Phylogenetic Analysis of Betanodavirus RNA2 Identified from Wild Marine Fish in Oceanic Regions

<>Takahiro Sakamoto1, Yasushi Okinaka1*, Koh-Ichiro Mori2, Takuma Sugaya2, Toyohiro Nishioka3, Masakazu Oka4, Hiroshi Yamashita5 and Toshihiro Nakai1<>1Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, 739-8528, Japan
2National Research Institute of Aquaculture, Fisheries Research Agency, Saiki, Oita 879-2602, Japan
3National Research Institute of Aquaculture, Fisheries Research Agency, Minami-Ise, Mie 516-0193, Japan
4Management Section of National Center for Stock Enhancement, Fisheries Research Agency, Yokohama, Kanagawa 220-6115, Japan
5Ehime Prefectural Fisheries Experimental Station, Uwajima, Ehime 798-0104, Japan
(Received May 1, 2007)
ABSTRACT唯etanodaviruses, the causative agents of viral nervous necrosis of fish in aquaculture, can be genetically classified into four types, designated SJNNV, RGNNV, TPNNV and BFNNV. To investigate betanodaviruses in apparently healthy wild fish, we tested 20 fish species from some surrounding ocean areas of Japan for the detection of viral RNA using PCR-based methods. Of 729 fish tested, six (six species) and 87 (11 species) samples were positive for RT-PCR and nested PCR, respectively. These positive species have never been reported as susceptible hosts. Sequencing of obtained PCR amplicons revealed that RGNNV and SJNNV existed in nine and six fish species, respectively. BFNNV was found from one species, round herring Etrumeus teres. Interestingly, the RNA2 sequences thus obtained had more variations than those from wild fish caught around aquaculture facilities in our previous report. These results suggest that genetically varied betanodaviruses are widely spread around Japan.

Key words: betanodavirus, viral nervous necrosis, coat protein gene, phylogenetic analysis

Limited Availability of PCR for the Detection of Renibacterium salmoninarum, the Causative Agent of Bacterial Kidney Disease

<>Ritsuko Okuda1,2, Toyohiko Nishizawa1 and Mamoru Yoshimizu1*<>1Faculty of Fisheries Sciences, Hokkaido University, Hakodate, 041-8611, Japan
2Japan Fisheries Resource Conservation Association, Tokyo 104-0054, Japan
(Received August 22, 2007)
ABSTRACT優etection limit of PCR was investigated for Renibacterium salmoninarum, the causative agent of bacterial kidney disease, with a logarithmic growth phase. When genomic DNAs from serially diluted bacterial cells were subjected to PCR, the minimum concentration by PCR-detection with 30, 35 and 40 cycles of amplification were all 105 cells/mL, and the sensitivity was not improved by treatments of the bacteria with lysozyme, achromopeptidase and/or SDS. The extraction rate of genomic DNAs from the bacterial cells was approximately 3%. It was confirmed that PCR-detection limit for R. salmoninarum cells mixed with fish kidney tissues was significantly lower than that for the bacteria suspended in the medium. Moreover, there was no significant difference in the detection limit between immunofluorescence antibody technique (IFAT) and PCR. These results suggest that PCR detection should be followed by other methods such as IFAT.

Key words: Renibacterium salmoninarum, bacterial kidney disease, BKD, PCR, IFAT

Novel Exophiala Infection Involving Ulcerative Skin Lesions in Japanese Flounder Paralichthys olivaceus

<>Osamu Kurata1*, Chutharat Munchan1, Shinpei Wada1, Kishio Hatai1, Yasuyuki Miyoshi2 and Yutaka Fukuda2<>1Department of Veterinary Science, Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602, Japan
2Fisheries Research Institute, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Saiki, Oita 879-2602, Japan
(Received September 26, 2007)
ABSTRACT輸 dematiaceous fungus was isolated from Japanese flounder Paralichthys olivaceus with ulceration and erosion of the skin surface. The fungus was identified as an Exophiala species, with different morphological, biological and molecular characteristics from three previously described pathogenic Exophiala species. Fungal hyphae extended laterally in the dermis, and were absent from the epidermis and musculature of the skin lesions and kidneys of the diseased fish. An inflammatory response with granuloma occurred in the dermis involving accumulations of epithelioid cells around the hyphae. The granulomas were surrounded by lymphocyte-like cells. Epidermal degeneration was observed above the inflamed dermis, suggesting that the inflammatory response caused epidermal damage. Experimental infection reproduced hyphal extension and infiltration of inflammatory cells in the dermis of the flounder, confirming the pathogenicity of the fungus.

Key words: Exophiala, fungal infection, Paralichthys olivaceus, dematiaceous fungus, ulceration, granuloma, epithelioid cell, dermis

Myxosporean Emaciation Disease of Cultured Red Sea Bream Pagrus major and Spotted Knifejaw Oplegnathus punctatus

<>Tetsuya Yanagida1*, Oswaldo Palenzuela2, Tatsumu Hirae3, Shinji Tanaka4, Hiroshi Yokoyama1** and Kazuo Ogawa1<>1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
2Instituto de Acuicultura de Torre de la Sal (CSIC), 12595 Ribera de Cabanes, Castellon, Spain
3Kagoshima Prefectural Fisheries Technology and Development Center, Ibusuki, Kagoshima 891-0315, Japan
4Mie Prefectural Science and Technology Promotion Center, Shima, Mie 517-0404, Japan
(Received November 16, 2007)
ABSTRACT柚yxosporean emaciation disease of cultured red sea bream Pagrus major and spotted knifejaw Oplegnathus punctatus has recently occurred in Japan. Morphological features and molecular analysis of SSU rDNA indicated that myxozoans from the intestine of affected fishes were identified as Enteromyxum leei, one of the causative organisms of the emaciation disease of tiger puffer Takifugu rubripes. A one-year periodic examination of E. leei infection in cultured red sea bream revealed that cumulative mortalities reached about 10% during the first summer, but surviving fish were not infected in the following year. Experimental transmission of E. leei from infected tiger puffer to naive red sea bream was achieved by both cohabitation with infected fish and exposure to effluent from a tank containing infected fish. This study suggests that fish-to-fish transmission occurs among different fish species in culture fields.

Key words: Myxozoa, Enteromyxum leei, emaciation disease, Pagrus major, Oplegnathus punctatus, Takifugu rubripes

A New Vibriosis of Cultured Ayu Plecoglossus altivelis

<>Takahiro Nagai1,2*, Yoshisuke Iida1, Emi Iwamoto2 and Toshihiro Nakai2<>1Fisheries and Marine Technology Research Center, Hiroshima Prefectural Technology Research Institute, Kure, Hiroshima 737-1207, Japan
2Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, 739-8528, Japan
(Received December 19, 2007)
ABSTRACT輸 mass mortality of cultured ayu Plecoglossus altivelis occurred from May to June in 2006 in Hiroshima Prefecture. Most of the diseased fish showed exophthalmos, reddening of the head and curvature of the body. A Gram-negative bacterium was isolated from the brain and kidney of affected fish by using a blood agar medium, but not by non-blood agars. The bacterium was well-cultivable on non-blood agars supplemented with hemin at 25 μg/mL or higher concentrations. The bacterium was classified into the genus Vibrio but differentiated from any known vibrios, based on the results of conventional characterization tests and sequence analyses of 16S rDNA and the non-coding region of the intergenic transcribed spacers between 16S and 23S rDNA. Intraperitoneal injection confirmed the pathogenicity of the bacterium to ayu (mean body weight: 14.8 g) with the LD50 at 1.4 × 103 CFU/fish. Bacteriological and histopathological examinations on the experimentally infected fish revealed that the causative bacterium has high affinity to the brain of ayu.

Key words: Plecoglossus altivelis, vibriosis, Vibrio sp., hemin, pathogenicity, ayu

Infection with the Parasitic Copepod Clavella parva (Lernaeopodidae) in Gold-eye Rockfish Sebastes thompsoni Broodstock in Japan

<>Kazuya Nagasawa1*, Tetsuya Umino1, Daisuke Uyeno2, Susumu Ohtsuka2 and Hiroaki Koizumi3<>1Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
2Takehara Marine Science Station, Setouchi Field Science Center, Graduate School of Biosphere Science, Hiroshima University, Takehara, Hiroshima 725-0024, Japan
3Aquaculture Institute, Aomori Prefectural Fisheries Research Center, Hiranai, Aomori 039-3381, Japan
(Received December 26, 2007)
ABSTRACT悠nfection with the lernaeopodid copepod Clavella parva was found in gold-eye rockfish Sebastes thompsoni broodstock kept at an aquaculture institute in Aomori Prefecture, northern Japan. A description of female and male adults of C. parva is given. This is the first record of C. parva from Japan and a new host record. The parasite was likely introduced to the institute by the fish that had been reared in cages kept in natural waters. No infection was observed again after manual removal of the copepods. The species occurred most abundantly on the dorsal and caudal fins, followed by the anal fin. Literary information on the geographical distribution and hosts of C. parva is reviewed.

Key words: Clavella parva, Sebastes thompsoni, parasitic copepod, new host, new locality