Bo LİU, Jinjuan WAN, Xianping GE, Jun XIE, Qunlan ZHOU, Linghong MIAO, Mingchun REN, Liangkun PAN

Effects of Dietary Vitamin C on the Physiological Responses and Disease Resistance to Ph Stress and Aeromonas Hydrophila Infection of Megalobrama Amblycephala

We evaluated the effect of vitamin C (Vit C) supplementation on the resistance of Megalobrama amblycephala to Aeromonas hydrophi1a infection under pH stress. Fish were randomly divided into six groups: a control group (fed with a basal diet) and five treatment groups (fed with basal diet supplemented with 33.4, 65.8, 133.7, 251.5 and 501.5 mg/kg Vit C, respectively). After 90 days, fish were exposed to combined stressors, first pH 9.5 followed by A. hydrophila infection. The results showed that 133.7 mg/kg vit C improved complement 4 (C4), anti-superoxide anion free aradical (ASAFER) and heat shock protein (HSP) 70 compared with the control group; and 251.5 mg/kg vit C enhanced complement 3 (C3), HSP60, HSP70 and HSP90 compared to the control group before stress. After pH stress and A. hydrophila infection, hemoglobin, ASAFER and HSP60, HSP70, HSP90 in the groups fed with 133.7 and 251.5 mg/kg vit C were still significantly higher, while serum cortisol in the group fed with 251.5 mg/kg vit C was lower compared to the control group 15 days after pH stress. The cumulative mortality of the control group was higher than that of the five treatment groups at 12, 24 h after A. hydrophila infection. The results of this study suggest that 133.7- 251.5mg/kg vit C in a diet could have potential to stimulate immune response, and enhance resistance against high pH stress and A. hydrophila infection of Wuchang bream.

PDF

___

Ai, Q.H., Mai, K.S., Tan, B.P., Xu, W., Zhang, W.B.,Ma, H.M., Liufu, Z.G. 2006. Effects of dietary vitamin C on survival, growth, and immunity of large yellow croaker, Pseudosciaena crocea. Aquaculture, 61:327- 336. doi:10.1016/j. aquaculture.2006.07.027

Aksoy, N., Vural, H., Sabuncu, T., Arslan, O., Aksoy, S. 2005. Beneficial effects of vitamins C and E against oxidative stress in diabetic rats. Nutrition Research, 25:625-630. doi:10.1016/j.nutres.2005.05.005

Barrosa., M.M., Falcon, D.R., de Oliveira, Orsi. R., Pezzato, L.E., Fernandes, Jr A.C., Guimarães, I.G., Fernandes Jr, A., Padovani, G.R., Sartori, M.M.P. 2014. Nonspecific immune parameters and physiological response of Nile tilapia fed ?-glucan and vitamin C for different periods and submitted to stress and bacterial challenge. Fish Shellfish Immun, 39:188- 195. doi: 10.1016/j.fsi.2014.05.004

Basu, N., Todgham, A.E., Ackerman, P.A., Bibeau, M.R., Nakano, K., Schulte, P.M., Iwama, G.K. 2002. Heat shock protein genes and their functional significance in fish. Gene, 295:173- 183. doi:10.1016/S0378-1119(02)00687-X

Boshra, H., Li, J., Sunyer, J.O. 2006. Recent advances on the complement system of teleost fish. Fish Shellfish Immun, 20:239-262. doi:10.1016/j.fsi.2005.04.004

Cellura, C., Toubiana, M., Parrinello, N., Roch, P.2006. HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus. Dev Comp Immun, 30:984-997. doi:10.1016/j.dci.2005.12.009

Chen, R.G., Lochmann, R., Goodwin, A., Praveen, K., Dabrowski, K., Lee, K.J. 2003. Alternative complement activity and resistance to heat stress in golden shiners (Notemigonus crysoleucas) are increased by dietary vitamin C levels in excess of requirement for prevention of deficiency signs. J Nutr, 133:2281-2286.

Cheng, W., Chen, J.C.2000. Effects of pH, temperature and salinity on immune parameters of the freshwater prawn Macrobrachium rosenbergii. Fish Shellfish Immun, 10:387-391. doi:10.1006/fsim.2000.0264

Cheng, W., Chen, J.C., 1998. Enterococcus-like infections in Macrobrachium rosenbergii are exacerbated by high pH and temperature but reduced by low salinity. Dis Aquat Org, 34:103-108. doi: 10.3354/dao034103

Cheng, W., Chen, S.M., Wang, F.I., Hsu, P.I., Liu, C.H., Chen, J.C. 2003. Effects of temperature, pH, salinity and ammonia on the phagocytic activity and clearance efficiency of giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae. Aquaculture, 219: 111-121. doi:10.1016/S0044-8486(03)00017-6

Cui, S.L., Liu, B., Xu, P., Xie, J., Wan, J.J., Zhou, M. 2013. Effects of emodin on growth, haemotological parameters and HSP70 mRNA expression of Wuchang bream (Megalobrama amblycephala) at two temperatures. Acta Hydrobilogyica Sinica., 37:919- 928.

Deane, E.E., Li, J., Woo, N.Y., 2004.Modulated heat shock protein expression during pathogenic Vibrio alginolyticus stress of sea bream. Dis Aquat Organ, 62: 205-215. doi: 10.3354/dao062205

Drape,H.H., Squires, E.J., Mahmoodi, H., Wu, J., Agarwal, S., Hadley, M. 1993. A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radical Bio Med, 15: 353-363.doi: 10.1016/0891- 5849(93)90035-S

Emata, A.C., Borlongan, I.G., Damaso, J.P. 2000. Dietary vitamin C and E supplementation and reproduction of milkfish Chanos chanos Forsskal. Aquac Res, 31:557-564. doi: 10.1016/0891-5849(93)90035-S

Eo, J., Lee, K.L.2008. Effect of dietary ascorbic acid on growth and non-specific immune responses of tiger puffer, Takifugu rubripes. Fish Shellfish Immun, 25:611-616. doi: 10.1016/j.fsi.2008.08.009

Gao. Q., Zhao, J., Song, L., Qiu, L., Yu, Y., Zhang, H., Ni, D. 2008. Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians. Fish Shellfish Immun., 24:379-385. doi: 10.1016/j.fsi.2010.10.019

Granelli, K., Bjorck, L., Appelqvist, L.A. 1995. The variation of superoxide dismutase (SOD) and xanthine oxidase (XO) activities in milk using an improved method to quantitate SOD activity. J Sci Food Agric, 67:85-91.

Ha, C.X., Liu, P., He,Y.Y., Li, J., Li, X. 2009. Effects of high pH on immune enzymes of "Huanghai No.1" population of shrimp Fenneropenaeus chinensis. J Fishery Sci China, 16:303-306.

Han, X.B., Xie, J.Z., Ba, C.F., Wang, G.C. 2011. Effect of dietary vit C on the hepatic the expression levels of HSP70 mRNA of rat. Shandong Medicine, 51:42-44.

He, L.J., Liao, L.K., Yuan, J.F., Tang, H.Y., Wu, Q., Zhang, G.W., 2006. Pathological observation of bacterial septicemia in Megalobrama amblycephala. J Southwest Univ, 3: 483-490.

Janda, J.M., Abbott, S.L. 1998. Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations, and unanswered questions. Clin Infect Dis, 27:332-344. doi: 10.1086/514652

Kong, X.H., Wang, G.Z., Ai, C.X., Li, S.J. 2004. Comparative study on content of reactive oxygen species and activity of antisuperoxide anion free radical in different organs and tissues o f mud crab, Scylla serrata. Marine Sciences, 28:1-4.

Kozinska, A., Figueras, M.J., Chacon, M.R., Soler, L.2002. Phenotypic characteristics and pathogenicity of Aeromonas genomospecies isolated from common carp (Cyprinus carpio L.). J Appl Microbiol, 93:1034- 1041. doi: 10.1046/j.1365-2672. 2002.01784.x

Kumari, J., Sahoo, P. K. 2005. High dietary vitamin C affects growth, non-specific immune responses and disease resistance in Asian catfish, Clarias batrachus. Mol Cell Biochem, 280: 25-33. doi: 10.1007/s11010- 005-8011-z

Lavilla-Pitogo, C.R., Leano, E.M., Paner, M.G. 1998. Mortalities of pond-cultured juvenile shrimp,Penaeus monodon, associated with dominance of luminescent vibriosis in the rearing environment. Aquaculture 164: 337-349. doi: 0.1016/S0044-8486(98)00198-7

Le Moullac, G., Haffner, P. 2000. Environmental factors affecting immune responses in crustacea. Aquaculture, 191: 121-131. doi: 10.1016/S0044-8486 (00)00422-1

Lee, K.J., Dabrowski, K., 2004. Long-term effects and interactions of dietary vitamins C and E on growth and reproduction of yellow perch, Perca flavescens. Aquaculture, 230:377-389. doi: 10.4172/2155- 9546.1000135

Li, C.C., Chen, J.C. 2008. The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under low and high pH stress. Fish Shellfish Immun, 25:701-709. doi:10.1016/j.fsi.2008.01.007

Lin, Y.C., Tayag, C.M., Huang, .CL., Tsui, W.C., Chen, J.C. 2010. White shrimp Litopenaeus vannameithat had received the hot-water extract of Spirulina platensis showed earlier recovery in immunity and upregulation of gene expressions after pH stress. Fish Shellfish Immun, 29: 1092-1098. doi:10.1016/j.fsi.2009.02.025

Lindquist, S., Craig, E.A.1998. The heat shock proteins. Annu Rev Genet, 22: 631-677. doi: 10.1146/annurev.genet.22.1.631

Liu, B., Ge, X.P., Xie, J., Xu, P., Cui, Y.T., Ming, J.H., Zhou, Q.L., Pan, L.K., 2012. Effects of anthraquinone extract from Rheum officinale Bail on the physiological responses and HSP70 gene expression of Megalobrama amblycephala under Aeromonas hydrophila infection. Fish Shellfish Immun, 32:1-7. doi: 10.1016/j.fsi.2011.02.015

Liu, B., Xie, J., Ge, X.P., Xu, P., Wang, A.M., He, Y.J., Zhou, Q.L., Pan, L.K., Chen, R.L. 2010. Effects of anthraquinone extract from Rheum officinale Bail on the growth performance and physiological responses of Macrobrachium rosenbergii under high temperature stress. Fish Shellfish Immun, 29:49-57. doi: 10.1016/j.fsi.2010.02.018

Ming, J.H., Xie, J., Xu, P., Ge, X.P., Liu, W.B., Ye, J.Y. 2012. Effects of emodin and vitamin C on growth performance, biochemical parameters and two HSP70s mRNA expression of Wuchang bream (Megalobrama amblycephala Yih) under high temperature stress. Fish Shellfish Immun, 32:651-661. doi: 10.1016/j.fsi.2012.01.008

Ministry of Agriculture of the People's Republic of China, 2013. Chinese Fisheries Yearbook. Chinese Agricultural Press, Beijing. Monfort, P., Baleux, B. 1990. Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a sewage treat-ment pond. Appl Environ Microbiol, 56:1999-2006

Monteroa, D., Marrerob, M., Izquierdoc, M.S., Robainac, L., Vergarac, J.M., Tortd, L. 1999. Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture, 269-278. doi: 10.1016/S0044-8486(98)00387-1

Nayak, S.K., Swain, P., Mukherjee, S.C.2007. Effect of dietary supplementation of probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita (Ham.). Fish Shellfish Immun, 23:892-896. doi:10.1016/j.fsi.2007.02.008

Ndong, D., Chen, Y.Y., Lin, Y.H., Vaseeharan, B., Chen, J.C., 2007. The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures. Fish Shellfish Immun, 22: 686-694. doi: 10.1016/j.fsi.2006.08.015

Ogara, W.O., Mbuthia, P.G., Kaburia, H.F.A., Sorum, H., Kagunya, D.K., Nduthu, D.I., Colquhoun, D.1998. Motile aeromonads associated with rainbow trout (Oncorhynchus mykiss) mortality in Kenya. Bull Eur Assoc Fish Pathol, 18:7-9.

Ojolick, E.J., Cusack, R., Benfey, T.J., Kerr, S.R. 1995. Survival and growth of all-female diploid and triploid rainbow trout (Oncorhynchus mykiss) reared at chronic high temperature. Aquaculture, 131: 177-187. doi: 10.1016/0044-8486(94)00338-O

Ortuno, J., Esteban, M.A., Meseguer, J.2002. Lack of effect of combining different stressors on immune responses of seabream (Sparus aurata L.). Vet Immunol Immunopathol, 84:17-27. doi:10.1016/S0165- 2427(01)00387-7

Ortuno, J., Esteban, M.A., Meseguer, J.2003. The effect of dietary intake of vitamins C and E on the stress response of gilthead seabream (Sparus aurata L.). Fish Shellfish Immun, 14:145-156. doi:10.1006/fsim.2002.0428

Özkan, F., Gündüz, S.G., Berköz, M., Hunt, A.Ö., Yalm, S.2012. The protective role of ascorbic acid (vitamin C) against chlorpyrifos-induced oxidative stress in Oreochromis niloticus. Fish Physiol Biochem, 38:635-643. doi: 10.1007/s10695-011-9544-6

Pan, L.Q., Zhang, L.J., Liu, H.Y. 2007. Effects of salinity and pH on ion-transport enzyme activities, survival and growth of Litopenaeus vannamei postlarvae. Aquaculture,273:711-720. doi:10.1016/j.aquaculture.2007.07.218

Qu, M., Shi, X.F., Zhang, Z.W., Ding, S.X. 2011. Cloning of HSP60 gene from Epinephelus akaara and its express characterization before and after vibrionic stressed. Acta Oceanologica Sinica, 33: 111-120.

Rotlant, J., Pavlidis, M., Kentouri, M., Abad, M.E., Tort, L.1997. Non-specific immune responses in the red porgy Pagrus pagrus crowding stress. Aquaculture, 156:279-290. doi:10.1016/S0044-8486(97)00075-6

Rungrassamee, W., Leelatanawit, R., Jiravanichpaisal, P., Klinbunga, S., Karoonuthaisiri, N. 2010. Expression and distribution of three heat shock protein genes under heat shock stress and underexpos ure to Vibrio harveyi in Penaeus monodon. Dev Comp Immunol, 34:1082-1090. doi:10.1016/j.dci.2010.05.012

Sobhana, K.S., Mohan, C.V., Shankar, K.M. 2002. Effect of dietary vitamin C on the disease susceptibility and inflammatory response of mrigal, Cirrhinus mrigala (Hamilton) to experimental infection of Aeromonas hydrophila. Aquaculture, 207: 225-238. doi:10.1016/S0044-8486(01)00793-1

Steinhagen, D., Kruse, P., Ko¨rting, W. 1989. Effects of immunosuppressive agents on common carp infected with the hemoflagellate Trypanoplasma borreli. Dis Aquat Organ, 7: 67-70. doi: 10.3354/dao007067

Tang, Y.K., Jia, Y.Y. 2008. The processing method study of real-time PCR data. Biotechnology, 18: 89-91.

Tewary, A., Patra, B.C. 2008. Use of vitamin C as an immunostimulant. Effect on growth, nutritional quality, and immune response of Labeo rohita (Ham.). Fish Physiol Biochem, 34:251-259. doi: 10.1007/s10695-007-9184-z

Topi?, Popovic., N., Teskeredzi?, E., Strunjak-Perovi?, I., Coz-Rakovac, R. 2000. Aeromonas hydrophila isolated from wild freshwater fish in Croatia. Vet Res Commun, 24:371-377.doi: 10.1023/A:1006418116155

Tort, L., Gomez, E., Montero, D., Sunyer, J.O. 1996. Serum haemolytic and agglutinating activity as indicators of fish immunocompetence. Their suitability in stress and dietary studies. Aquaculture Int, 4:31-41. doi: 10.1007/BF00175219

Trip, R.A., Maule, A.G., Schreck, C.B., Kaattari, S.L. 1987. Cortisol mediated suppression of Salmonid lymphocyte responses in vitro.Dev. Comp.Immunol, 11: 565-576. doi:10.1016/0145-305X(87)90045-0

Vivas, J., Carracedo, B., Riañ, J., Razquin, B.E., LópezFierro, P., Acosta, F., Naharro, G.J., Villena, A. 2004. Behavior of an Aeromonas hydrophila aroA live vaccine in water microcosms. Appl. Environ. Microbiol, 70: 2702-2708. doi: 10.1128/AEM.70.5.2702-2708.

Wan, J.J., Liu, B., Ge, X.P., Xie, J., Cui, S.L., Zhou, M.2013. Effects of dietary vitamin C on growth performance, hematology and muscle physiochemical indexes of juvenile Wuchang bream (Megalobrama amblycephala). J. Shanghai Ocean Univ., 22: 112- 119.

Wang, C., Silva, J.L. 1999. Prevalence and characteristics of Aeromonas species isolated from processed channel catfish. J Food Prot, 62:30-34. Wendelaar Bonga, S.E. 1997. The stress response in fish. Physiol Rev, 77: 591-625.

Wilkie, M.P., Wood, C.M. 1994. The effects of extremely alkaline water (pH 9.5) on rainbow trout gill function and morphology. J.Fish Biol, 45:87-98. doi: DOI: 10.1111/j.1095-8649.1994.tb01288.x

Yeh, S.T., Li, C.C., Tsuei, W.J., Chen, J.C.2010. The protective immunity of white shrimp Litopenaeus vannamei that had been immersed in the hot-water extract of Gracilaria tenuistipitata and subjected to combined stresses of Vibrio alginolyticus and temperature change. Fish Shellfish Immun, 29: 271- 278.doi: 10.1016/j.fsi.2010.04.014

Yin, Z., Lam, T.J., Sin, Y.M. 1995. The effects of crowding stress on the non-specific immune response in fancy carp (Cyprinus carpioL.). Fish Shellfish Immun, 5: 519-529. doi:10.1016/S1050-4648(95)80052-2

Zhou, M., Liu, B., Ge, X.P., Xie, J., Cui, Y.T., Wan, J.J., Cui, S.L.2013. Effects of Vitamin E on serum biochemical indexes and antioxidant capacity of Wuchang bream ( Megalobrama amblycephala) under acute high temperature stress and recovery. J Fisheries China, 37:117-125.