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NO38 - 2

Morphology and Immunological Roles of Hemocytes and Fixed Phagocytes in Black Tiger Shrimp Penaeus monodon

<>Kidchakan Supamattaya1*, Jareeporn Ruangsri1, Toshiaki Itami2, Vudthikorn Chitiwan1, Wutiporn Phromkunthong1 and Kiyokuni Muroga3<>1Aquatic Animal Health Research Center, Department of Aquatic Science,
Faculty of Natural Resources, Prince of Songkla University,
Had Yai, Songkhla 90112, Thailand
2Department of Biological Production and Environmental Science, Faculty of Agriculture,
Miyazaki University, Miyazaki 889-2192, Japan
3Graduate School of Biosphere Sciences, Hiroshima University,
Higashihiroshima 739-8528, Japan

(Received December 6, 2002)
ABSTRACT - Morphology and immunological roles of the three distinct types of hemocytes,
hyaline, small granular and large granular cells in black tiger shrimp Penaeus monodon were studied. The study of functions of these hemocytes in the elimination of injected yeast (Saccharomyces cerevisiae) or Vibrio harveyi as foreign bodies showed a rapid response against those particles. Together with fixed phagocytes, the blood cells removed the yeast through the process of phagocytosis, nodule formation and encapsulation, which occurred at almost all parts of the body. The injection of the yeast caused a marked reduction in the blood cell counts in the hemolymph during the first 1 h. V. harveyi was efficiently removed within 3 h after injection. The granular cells (small granular and large granular hemocytes) were the major blood cells that are associated with phenoloxidase activity. The role of hemocytes and fixed phagocytes on defense mechanism in the shrimp were discussed.

Key words: Penaeus monodon, hemocyte, fixed phagocyte, phagocytosis, immune parameter, phenoloxidase, black tiger shrimp

Effects of Water Temperature on the Development of the Monogenean Neoheterobothrium hirame on Japanese Flounder Paralichthys olivaceus

<>Nobuyuki Tsutsumi1, Tomoyoshi Yoshinaga1, Takashi Kamaishi2,Chihaya Nakayasu2 and Kazuo Ogawa1*<>1Laboratory of Fish Diseases, Department of Aquatic Bioscience, Graduate School of Agricultural
and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
2National Research Institute of Aquaculture, Fisheries Research Agency,
Nansei, Mie 516-0193, Japan

(Received December 9, 2002)
ABSTRACT - Development and growth of the monogenean Neoheterobothrium hirame on
Japanese flounder Paralichthys olivaceus were investigated at 15-C, 20-C and 25-C, and production of antibodies against N. hirame was monitored in relation to the development of the parasite. After settlement on the gill filaments, hematin cells in the gut wall of N. hirame were first observed on Day 17, Day 10 and Day 7 post-exposure (PE) of oncomiracidia at 15-C, 20-C and 25-C, respectively. The parasite started to move to the buccal cavity wall from Day 38, Day 24 and Day 24 PE and eggs were observed in the uterus from Day 59, Day 38 and Day 31 PE at each temperature, respectively. Intensity of infection decreased subsequently and infection persisted until Day 122, Day 66 and Day 52 PE, respectively. Antibody titers gradually increased from Day 52 PE at 15-C, while at 20-C and at 25-C, they rapidly increased after a decrease in the intensity and prevalence of the infections was observed. This suggests that the death of the parasite
induces a host reaction, resulting in the production of antibodies against N. hirame.

Key words: Neoheterobothrium hirame, Monogenea, water temperature, Paralichthys olivaceus, development

Histological Detection of Aquatic Fungi by Uvitex 2B,a Fluorescent Dye

<>Shinpei Wada*, Yamato Yorisada, Osamu Kurata and Kishio Hatai<>Division of Fish Diseases, Nippon Veterinary and Animal Science University,
Musashino, Tokyo 180-8602, Japan

(Received January 23, 2003)
ABSTRACT - Conventional staining methods for fungal detection are time consuming, technically demanding, and instable dye-affinity in different fungal species, especially in aquatic fungi. In this study, Uvitex 2B [4, 4-BIS (2-di (2-hydroxyethyl)-amino-4- (3-sulfophenylamino)-1, 3, 5- triazine-6-ylamino)-stilbene-2, 2・ disulfonic acid, sodium salt] was applied to detect fungal elements in paraffin sections of some aquatic animals. As a result, it was found that Uvitex 2B was superior to Gomori痴 methenamine-silver nitrate Grocott痴 variation, periodic acid-Schiff reaction and Schmorl痴 method for staining aquatic fungi in tissue sections. In addition, Uvitex 2B required much shorter time and less specialized skills in the staining procedure than Grocott. Although it has been known that oomycete fungi are difficult to be detected in histopahological sections because of their
unstable stainabilities with other staining methods, Uvitex 2B provided excellent results to detect them in tissues of aquatic animals.

Key words: detection, aquatic fungi, Uvitex 2B, histology, fluorescence

Odontoma in an Elkhorn Sculpin Alcichthys elongatus Caught in the Sea of Japan

<>Yoshiharu Honma*, Jiro Hitomi, Masaei Takeda and Tatsuo Ushiki<>Division of Microscopic Anatomy and Bio-imaging, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan

(Received February 10, 2002)
ABSTRACT - A prominent odontoma was found projecting from the maxilla of an elkhorn sculpin Alcichthys elongatus, 27.4 cm in total length, captured in a trap for boreal whelk set offshore from Toyama Bay, the Sea of Japan, in late June, 2002. Microscopic examination of large spherical tumor (29 mm in diameter, 19 mm in depth and 6.5 g in weight) protruding well outside the mouth cavity revealed it to be surrounded by squamous epithelium containing numerous dental tissue masses (imperfect teeth or teeth germ) throughout the stromal tissue. The teeth germ comprised odontoblasts, predentin and calcified dentin. No enamel (substantia adamantia) was demonstrated, although several fragments of calcified trabecular (spongy) bones were encountered. This type of tumor, diagnosed as a compound odontoma, seems to be the first report of such from a teleost from Asian-Oceanic waters. The cause of the tumor is unknown.

Key words: odontoma, fish tumor, Alcichthys elongatus, tooth, elkhorn sculpin, Sea of Japan

Mortality in Pond-Cultured Shrimp Penaeus monodon in the Philippines Associated with Vibrio harveyi and White Spot Syndrome Virus

<>Leobert D. de la Pe紡1*, Celia R. Lavilla-Pitogo1, Atsushi Namikoshi2, Toyohiko Nishizawa3, Yasuo Inui1 and Kiyokuni Muroga2<>1Fish Health Section, SEAFDEC Aquaculture Department, Tigbauan, Iloilo, Philippines
2Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
3Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan

(Received October 4, 2002)

ABSTRACT - Heavy mortalities were observed among pond-cultured Penaeus monodon in the provinces of Bohol, Misamis Occidental, Lanao del Norte and Zamboanga del Sur, Philippines. Vibrio harveyi was isolated purely from the hepatopancreas and lymphoid organs of affected shrimp and histopathological observations indicated a severe bacterial infection in the shrimp. Majority of the samples gave negative results in the one-step PCR for the detection of white spot syndrome virus (WSSV). However, nested PCR produced amplicons specific for WSSV DNA from most of the shrimp tested. These results suggest that shrimp were infected dually with V. harveyi and WSSV, but the major causative agent of the present mortalities was V. harveyi.

Key words: Penaeus monodon, Vibrio harveyi, WSSV, mixed infection, luminescent vibriosis, white spot syndrome

Field Trials of a Vaccine with Water-soluble Adjuvant for Bacterial Coldwater Diseasein Ayu Plecoglossus altivelis

<>Takahiro Nagai, Yoshisuke Iida and Takashi Yoneji<>Hiroshima Prefectural Fisheries Experiment Station, Ondo, Hiroshima 737-1207, Japan

(Received January 6, 2003)

ABSTRACT - The efficacy of a vaccine with water-soluble adjuvant (Montanidae IMS 1312)for bacterial coldwater disease was examined in ayu Plecoglossus altivelis under field conditions. Ayu injected intraperitoneally with formalin-killed bacterin of Flavobacterium psychrophilum mixed with the water-soluble adjuvant were reared in concrete ponds with river water from
April to September in 2000 and 2001. Coldwater disease
occurred spontaneously from the middle of May
to the middle of June (2000) and to the middle of July (2001). The mortalities of twice-vaccinated ayu were significantly lower than non-vaccinated ayu in both
years and relative percent survivals (RPS) were 33.0% in 2000 and 39.6% in 2001.

Key words: coldwater disease, Plecoglossus altivelis, vaccination, water-soluble ajuvant, Flavobacterium psychrophilum, field trial, ayu

Effectiveness of Fasting against Red Sea Bream Iridoviral Disease in Red Sea Bream

<>Shinji Tanaka*, Hideo Aoki1, Misa Inoue and Isao Kuriyama<>Owase Fisheries Laboratory, Fisheries Research Division, Mie Prefectural Science and Technology Promotion
Center, Owase, Mie 519-3602, Japan
1Present address:Fisheries Research Division, Mie Prefectural Science and Technology Promotion
Center, Hamajima, Mie 517-0404, Japan

(Received March 20, 2003)

ABSTRACT - Effectiveness of fasting against red sea bream iridoviral disease (RSIVD) in juvenile red sea bream Pagrus major were evaluated. In an infection
experiment, groups which were fasted for 10 or 20 days after challenge showed significantly lower mortalities than a feeding group. In a field trial test, in which a naturally RSIVD-prevailing batch of red sea bream was divided into 2 groups, a fasting group showed significantly lower mortality than a feeding group. These results indicate the effectiveness of fasting after virus infection against RSIVD.

Key words: red sea bream iridoviral disease, fasting, Pagrus major, iridovirus, RSIVD