P-274. Molecular cloning, expression analysis and gene knockdown of cata- lase gene from kuruma shrimp Marsupenaeus japonicus D. Shigeyoshi 1 , M. Inada 2 , T. Kono 3 , T. Yoshida 1 , M. Sakai 1 , T. Itami 1, * . 1 Faculty of Agriculture, University of Miyazaki, Japan; 2 Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, Japan; 3 Interdisciplinary Research Organization, University of Miyazaki, Japan Abstract Nitric oxide (NO) and reactive oxygen species (ROS) are known as free radicals. The ROS superoxide (O2-) and the superoxide-derived hydrogen peroxide (H2O2) are predominantly associated with antibacterial and antiviral host defense mechanisms. The catalase gene is the key gene coding for the enzyme splitting hydrogen peroxide into water and oxygen. In the present study, we describe the entire cDNA sequence (1,826 bp) of Marsupenaeus japonicus (kuruma shrimp) catalase (MjCat). The open reading frame of MjCat encodes a protein of 520 amino acids with an estimated mass of 58 kDa. In analyses using amino acids sequences, MjCat was found to be closely related to crustacean catalase and has 94.4% sequence homology with the Cat gene of the freshy prawn, Fenneropenaeus chinensis. In gene expression analysis, MjCat mRNA is highly expressed in the lymphoid organ, hepatopancreas and stomach of the healthy kuruma shrimp. In MjCat gene knockdown experiment, the survival rates signifi- cantly decreased over 7 days after gene knockdown treatment. M. Inada is a recipient of the Japan Society for the Promotion of Science (JSPS). This study was supported, in part, by research grants from the JSPS and the JSPS Asian CORE Program. * Corresponding author. E-mail address: itamit@cc.miyazaki-u.ac.jp (T. Itami) P-438. A comparison of immunoglobulin M (IgM) from Pangasius-related species and their reaction with anti-Pangasius hypophthalmus IgM monoclonal antibodies W. Sirimanapong 1, 2, * , K.D. Thompson 1 , K. Kledmanee 2 , P. Thaijongrak 2 , B. Collet 3 , E.L. Ooi 4 , O. Byron 5 , A. Adams 1 . 1 Institute of Aquaculture, University of Stirling, Stirling, Scotland, United Kingdom; 2 Faculty of Veterinary Sciences, Mahidol University, Salaya campus, Nakornpathom, Thailand; 3 Marine Scotland-Science, Scottish Government, Marine Laboratory, Aberdeen, Scotland, United Kingdom; 4 Novus International, Novus Aqua Research Center, Linh Trung Ward, Thu Duc District, Ho Chi Minh, Vietnam; 5 School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Scotland, United Kingdom Abstract Pangasianodon hypophthalmus immunoglobulin M (IgM) was purified from plasma by affinity chromatography and sodium sulphate precipitation (14, 16 and 20%). Sedimentation equilibrium analysis using analytical ul- tracentrifugation was performed to determine the molecular weight of the native molecule. The IgM of other Asian species Pangasianodon gigas, Pangasius larnaudii, Pangasius sanitwongsei, Pangasius borcoti, Hamibragus filamentus, Hamibargus wyckioides, Clarias bactracus, Clarias macrocephalus and Cyprinus carpio were prepared using 14% sodium sulphate precipita- tion. Purified IgM was analysed using sodium dodecyl sulphate-poly- acrylamide gel electrophoresis (SDS-PAGE, 12.5%) and the results indicated that the heavy and light chains of IgM from P. hypophthalmus were 70- 72kDa and 25-26 kDa, respectively. The light (L) chains of IgM from the other fish species were similar to P. hypophthalmus, while the heavy (H) chains varied (P. gigas and P. larnaudii 76kDa, P. sanitwongsei 69kDa, H. fil- amentus 73kDa, P. borcoti and H. wyckioides 75kDa, C. bactracus 74kDa, C. macrocephalus 73kDa and C. carpio 70kDa), as did the native IgM molecules. Western blot analysis of the IgM from the different fish species, performed using six anti-P. hypophthalmus IgM monoclonal antibodies (mAbs) pro- duced against affinity purified IgM, showed that two of the mAbs (mAbs 23 and 28) reacted with the heavy chains of the IgM molecule for all the fish species tested. The other mAbs (mAbs 1, 2, 7 and 18) reacted with the light chains of most species tested, with the exception of mAbs 2 and 7 which did not react with the C. carpio light chains. The serum antibody response of P. hypophthalmus, experimentally infected with Edwardsiella ictaluiri by immersion, was successfully measured by an enzyme-linked immuno- sorbent assay using mAb 23. * Corresponding author. E-mail address: wanna.sirimanapong@stir.ac.uk (W. Sirimanapong) P-232. Evaluation of citrus by-product on growth performance, innate im- munity and disease resistance against Edwardsiella tarda of Korean rockfish Sebastes schelgeli J.W. Song, K.J. Lee * . Faculty of Marine Biomedical Sciences, Jeju National University, Jeju 690-756, Korea Abstract Effects of dietary vitamin C source on growth performance, feed utilization, innate immunity and disease resistance of Korean rockfish were evaluated. A basal diet without vitamin C was regarded as a control and four other diets were formulated to contain L-ascorbyl-2-polyphosphate, citrus by-product (CBP) and fermented CBP by probiotic bacteria, Bacillus subtilis or B. pumilus, at a level of 100 mg ascorbic acid equivalent/kg diet (designated as Con, LAPP, CBP, BS-CBP and BP-CBP, respectively). Korean rockfish (body weight, 24.1 g) were fed the five experimental diets to apparent satiation for 13 weeks. Growth performance and feed utilization were significantly improved in fish fed the CBP and BP-CBP diets compared to those of fish fed the Con diet. During 25 days of a challenge test, fish fed vitamin C-supplemented diets had significantly higher survival rates (67%, 67%, 71% and 67%) than fish fed the Con diet (40%) against Edwardsiella tarda. The results indicate that CBP or fermented CBP with Bacilius sp. has great potential as an alternative vitamin C source in diets for Korean rockfish without any adverse effects. More data on non-specific immune responses will be presented in detail. * Corresponding author. E-mail address: kjlee@jejunu.ac.kr (K.J. Lee) P-163. Monoclonal antibodies recognizing serum immunoglobulins and sur- face immunoglobulin-positive cells of Catla catla (Hamilton, 1822) N. Sood * , D.K. Chaudhary, G. Rathore, P.K. Pradhan, P. Punia, J.K. Jena. Fish Health Management Division National Bureau of Fish Genetic Resources, Lucknow 226002, Uttar Pradesh, India Abstract Catla catla, commonly known as catla or bhakur, is the fastest growing In- dian major carp and an important aquaculture species in Southeast Asia. The serum immunoglobulins were purified from catla immunized with bovine serum albumin, using BSA-CL agarose affinity column. The reducing SDS- PAGE of purified Ig revealed two major bands with molecular weight (MW) of 90.8 and 27.2 kDA, corresponding to heavy (H) and light (L) chains, respectively. In non-reducing gradient SDS-PAGE, three bands with MW of 851.8, 480.7 and 249 kDA were observed. The purified immunoglobulins were used to produce two monoclonal antibodies, designated G10/1 and F9/ 2. Both the monoclonal antibodies were specific to H chain of reduced Ig and belonged to IgG1/k isotype. G10/1 MAb was evaluated for its application as a diagnostic reagent in a variety of immunoassays. In western blotting of non- reduced Ig, G10/1 MAb stained all the three bands observed in non-reducing SDS-PAGE, implying that they were the redox forms of Ig. The MAb did not show any reactivity with serum of Labeo rohita, Cirrhinus mrigala, Channa Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752 1738