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NO55 - 4

Hygiene Practices for Aquaculture and Hatchery Management

Hisae Kasai* and Jun Nagataa

Division of Marine Life Science, Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041-8611, Japan

(Received October 31, 2020)

ABSTRACT―Water supplies for seed production and aquaculture may also be pathways for the introduction and spread of infectious diseases. A pathogen free water source is essential for success in aquaculture. Water commonly used in aquaculture comes from coastal waters or rivers and may contain fish pathogens. Such open water supplies should not be used without prior treatment. Disinfection of wastewater before it is discharged is necessary to avoid contamination of the environment with pathogens. Disinfection may be done using ultraviolet (UV), oxidants produced by ozonation of seawater, or hypochlorite produced by electrolyzation of seawater for disinfection of water. Disinfection of eggs can effectively eliminate surface-borne pathogens. Iodophors appear to be the best disinfectant and offer a wide margin of safety to salmonid eggs. For marine fish eggs, ozonated or electrolyzed seawater are used as disinfectants. Moreover, general sanitation measures are standard practice in hatchery management and aquaculture. Methods used to sanitize a rearing unit should take into account chemical toxicity to fish and consequences of prolonged use.

Key words: fish, disease, hygiene, disinfection, UV, ozone, electrolyzation

Benedenia akajin n. sp. (Monogenea: Capsalidae) from Leopard Coral Grouper Plectropomus leopardus Reared in Okinawa Prefecture, Japan

Kazuo Ogawa1*, Hirotaka Mizuochi2, Tomofumi Yamaguchi3, Sho Shirakashi4, Nanami Asai4and Yasuo Agawa4

1 Meguro Parasitological Museum, Tokyo 153-0064, Japan
2 Stock Enhancement Center of Lake Biwa, Kusatsu, Shiga 525-0005, Japan
3 Yaeyama Field Station, Fisheries Technology Institute, National Research and Development Agency, Japan Fisheries Research and Education Agency, Okinawa 907-0451, Japan 4Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan

(Received April 29, 2020)

ABSTRACT―The grow-out stage of leopard coral grouper Plectropomus leopardus reared in land-based tanks in Okinawa Prefecture, Japan, were often heavily infected with capsalid monogeneans, up to a few millimeters in size, on the fins and skin. Morphological investigation revealed that the parasite belongs to Benedenia (Monogenea: Capsalidae). Benedenia akajin n. sp. is proposed here and described morphologically, based on the unique structures in the vaginal duct with a thick-walled distal one-third, the penis with a teat-shaped tip and the testes smaller than the germarium. A positive relationship between the lengths of three haptoral sclerites (anterior sclerites and anterior and posterior hamuli) and body length indicates that these taxonomically important structures keep growing as the parasite ages. Molecular analyses showed that the present species is most closely related to B. epinepheli and B. sargocentron, with the highest 28S rDNA identity of 98.1%, but no identical sequences were found in the database. Both morphological and molecular evidences suggest Benedenia akajin n. sp. as an undescribed species, and this new skin fluke deserves further investigations for the stable aquaculture of P. leopardus.

Key words: leopard coral grouper Plectropomus leopardus, capsalid monogeneans, Benedenia, 28S rDNA

5-aminolevulinic Acid's Effects on Immune-related Gene Expression and Acute Hepatopancreatic Necrosis Disease (AHPND) Protection in Pacific White Shrimp Litopenaeus vannamei

Sataporn Direkbusarakom1*, Hironobu Kinoshita2, Chettupon Pooljun1, Sudarat Saeng-ngern1 and Suwit Wuthisuthimethavee1

1 Research Center of Excellence for Shrimp, School of Agricultural Technology, Walailak University, Nakhon Si Thammarat 80160, Thailand
2 NEOPHARMA Japan Co., Ltd, Tokyo 102-0071, Japan

(Received May 14, 2020)

ABSTRACT―5-aminolevulinic acid (5-ALA) is an endogenous non-protein amino acid that is widely used in medicine, pharmacy products, agriculture, and aquaculture. In this study, the aim is to investigate the effects of 5-ALA on feeding Pacific white shrimp (Litopenaeus vannamei) with a diet supplemented with 5-ALA on their growth performance, immune responses, and resistance to acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio paraheamolyticus. Shrimp were fed with different diets, including a commercial diet (control) and diets supplemented with 15 ppm (ALA15) and 30 ppm (ALA30) of 5-ALA for 15 and 30 days before bacteria challenge. Anti-lipopolysaccharide factor, crustin, and penaeidin3 immune genes had significantly increased expression level after 15 and 30 days of feeding with 5-ALA. The mortality rate of L. vannamei fed with 5-ALA for 15 and 30 days significantly decreased after V. parahaemolyticus infection. These results indicated that the diet supplemented with 5-ALA enhanced the innate immune response and consequently shrimp's pathogen tolerance. Thus, 5-ALA can be used as immunostimulant additive for Pacific white shrimp cultivation.

Key words: 5-aminolevulinic acid, 5-ALA, L. vannamei, V. parahaemolyticus, AHPND, immune genes

Changes in Resistance against Bacterial Cold-Water Disease and in Leukocyte Composition along with Sexual Maturation in Ayu Plecoglossus altivelis

Nayu Kawashima1, Shungo Minami1, Kyuma Suzuki2, Shun Watanabe2, Chihaya Nakayasu3, Motohiko Sano1 and Goshi Kato1*

1 Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
2 Gunma Prefectural Fisheries Experiment Station, Gunma 371-0036, Japan
3 Pathology Division, Aquaculture Research Department, Fisheries Technology Institute, National Research and Development Agency, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan

(Received June 16, 2020)

ABSTRACT―In the present study, we investigated changes in resistance against bacterial cold-water disease and in leukocyte composition along with sexual maturation in ayu. Fish were reared under long photoperiod (LP) and the natural day length (ND) during September to November. Ayu sampled before starting the experiment was used as an initial control (IN). Gonad-somatic index significantly increased in ND group, but not in LP group, compared with IN group. Cumulative survival rate after challenge with 2.7 × 104 CFU of Flavobacterium psychrophilum was significantly higher in LP group (90%) than that in ND group (0%). Gene expression levels for lymphocyte markers (IgM, IgD, CD3ε and TCRβ) in the trunk kidney were significantly lower in ND group compared with the values in IN group. Consistently, the decrease of IgM and CD8α mRNA positive cells in the trunk kidney of ND group was also shown by in situ hybridization. Further, flow cytometry revealed that percentage of B lymphocytes in the trunk kidney leukocytes were significantly decreased in ND group than in the other groups. These data suggest that sexual maturation is the major cause of the decrease in disease resistance and of the lymphocytopenia, in ayu during the spawning season.

Key words: Lymphocytopenia, T lymphocyte, B lymphocyte, Thrombocyte, Plecoglossus altivelis, Flavobacterium psychrophilum, BCWD

Larval Cucullanid Nematode Infection in the Liver of Ayu

Mitsuru Okamoto1, Akira Okino1, Mark A. Freeman2 and Kazuo Ogawa 3*

1 Shimane Prefectural Fisheries Technology Center, Shimane 697-0051, Japan
2 Ross University School of Veterinary Medicine, St. Kitts, West Indies
3 Meguro Parasitological Museum, Tokyo 153-0064, Japan

(Received July 16, 2020)

ABSTRACT―Many white, elliptical to spherical cyst-like bodies (0.43-1.57 mm in diameter) were found in the liver of ayu Plecoglossus altivelis in Shimane Prefecture, Japan. Each cyst contained one or two (rarely three) nematodes, 1.11-1.20 mm in body length. In parasitological surveys of ayu conducted from 2008 to 2013, infection was confirmed in ayu from three out of six river systems in the prefecture, with prevalence and intensity of infection ranging widely from 0 to 100% and from 1 to 837 cysts/ fish, respectively. We believe that infection occurred in rivers, inferred from the fact that uninfected artificially produced ayu became infected after release to rivers. Morphologically, the nematodes were identified as third stage larvae. They stayed motionless inside the spherical bodies up to 24 h after capture of the host but started to move actively when exposed to physiological saline, where they survived for up to 81 h at room temperature of 22-25°C, <12 h at 37°C or <1 min at 80°C. Molecular phylogenetic analysis using 18S rDNA revealed that the nematode was robustly placed in the monophyletic Cucullanidae clade but could be identified as either Cucullanus or Dichelyne. The lifecycle remains unknown.

Key words: Cucullanidae, parasite, Plecoglossus altivelis, life cycle, third stage larva, nematode, food safety

Immunohistochemistry for Detection of MHC Class II-Expressing Cells in Japanese Flounder Paralichthys olivaceus

Osamu Kurata1*, Suyu Miyashita1, Shusei Toda1, Shinpei Wada1 and Takamitsu Sakai2

1 Laboratory of Aquatic Medicine, School of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan
2 Tamaki Field Station, Fisheries Technology Institute, National Research and Development Agency, Japan Fisheries Research and Education Agency, Mie 519-0423, Japan

(Received September 24, 2020)

ABSTRACT―Antigen-presenting cells characterised by the expression of MHC class II molecules play a crucial role in differentiating and activating helper T cells at lymphoid organs. We attempted to establish an immunohistochemical staining technique for the detection of MHC class II-expressing cells in Japanese flounder to study the localisation of these cells in tissues. We produced a specific antibody against the α chain peptide of MHC class II. The antibody reacted to the recombinant protein of the α2 segment of the class II molecule, the native molecule of the class II α chain in soluble proteins from the spleen and in membrane proteins from intraperitoneal infiltrated cells. Immunohistochemical staining using the antibodies could be utilised for the detection of MHC class II-expressing cells in paraffin-embedded tissue specimens. MHC class II-expressing cells were found in various organs and tissues such as the spleen, kidney, thymus, intestine, stomach, liver, heart, gills, skin, olfactory organs, and brain. The immune-positive cells showed an elongated- or rounded-shape. Considering our observations of morphological features and tissue localisation along with previously published information, we suggest that the MHC class II-expressing cells are dendritic cells, macrophages, B cells, microglia, and epithelial cells that have been considered antigen-presenting cells.

Key words: antigen-presenting cells, lymphoid organs, anti-MHCII α chain antibody, immunostaining, histology, immunoblotting, spleen, teleost

Seasonality of Clinostomum complanatum Cercariae in the Snail Host Radix auricularia japonica and Drug Treatment of the Parasite in Goldfish Carassius auratus

Jumpei Arakawa1*, Kohta Suzuki2 and Ryo Ishii3

1 Aichi Fisheries Research Institute, Aichi 443-0021, Japan
2 Yatomi Station, Aichi Fisheries Research Institute, Aichi 498-0017, Japan
3 Niigata Prefectural Inland Water Fisheries Experiment Station, Niigata 940-1137, Japan

(Received June 5, 2020)

ABSTRACT―Clear peaks of trematode Clinostomum complanatum cercariae detection rate in snails Radix auricularia japonica were observed around July in a goldfish Carassius auratus farm in Aichi Prefecture. Effects of Trichlorfon and praziquantel were examined against C. complanatum in artificially infected goldfish. Both were effective in the early phase of infection in goldfish cohabitating with infected snails. Trichlorfon was effective in a 300 mg/t bath treatment for 13 d and praziquantel was effective with 3 d of oral administration at 138 mg/kg body weight. Tratements with trichlorfon or praziquantel around July may reduce infection of C. complanatum without serious damage to fish caused by the excystation induced by the drug treatment in ornamental fish farms.

Key words: Seasonality, praziquantel, trichlorfon, deworming, Clinostomum, goldfish