Effect of dietary phosphorus level on defense system parameters and liver HSP70 concentration in juvenile Japanese flounder, Paralichthys olivaceus

Dietary phosphorus effects in Japanese flounder


  • Orhan UYAN Sciences of Marine Resources, The United, Graduate School of Agricultural Sciences (RENDAI), Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan https://orcid.org/0000-0002-3131-3849
  • Shunsuke KOSHIO
  • Manabu ISHIKAWA
  • Saichiro YOKOYAMA




Dietary Phosphorus Level, Mucus Lysozyme Activity, Mucus Bactericidal Activity, Liver HSP70s level, Growth, Paralichthys olivaceus


A feeding trail was performed to investigate the effect of dietary phosphorus (P) level on defense system in juvenile Japanese flounder. Calcium mono phosphate was used as dietary inorganic-P source. Three semi-purified diets with 3 levels of P (6, 10 and 18 g/kg diet, respectively) were prepared and fed to juveniles (average initial body weight, means ± S.E., 7.3 ± 0.3 g) for 30 days. After termination of the feeding trail, growth parameters were calculated, and mucus lysozyme activity, mucus bactericidal activity and the levels of heat shock protein 70 family (HSP70s) in liver were measured. Results indicated that final weight, weight gain and feed efficiency significantly improved with increasing dietary P level, and the highest growth parameters were obtained from the fish fed diet containing 18 g/kg P. Mucus lysozyme activity and mucus bactericidal activity were significantly improved with the increasing dietary P level. The highest level of HSP70s in liver (35.97 ng/mg protein) was observed on fish fed the diet containing 18g/kg P, and it was 2 and 2.6 folds higher (P<0.05) than those fish fed the diet with 10 and 6 g/kg P, respectively. In conclusion, P deficiency has significant effect on growth and measured defense system parameters, therefore, the dietary inclusion of P might affect the defense system and stress tolerance of juvenile Japanese flounder. Highest level of mucus lysozyme activity, HSP70s in liver, and bactericidal activities with reduced survival rate of E. coli was observed in fish fed diets incorporated with 1.8% P, that also maintained best growth performance in Japanese flounder.


A.O.A.C. (1990). Official Methods of Analysis of the Association of Official Analytical Chemists, 15th ed. Association of Official Analytical Chemists, Arlington, VA, USA.

Bierkens, J., Maes, J., & Plaetse, F.V. (1998). Dose-dependent induction of heat shock protein 70 synthesis in Raphidocelis subcapitata following exposure to different classes of environmental pollutants. Environmental Pollution, 101, 91-97. https://doi.org/10.1016/S0269-7491(98)00010-4

Bligh, E.G., & Dyer, W.J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911-917. https://doi.org/10.1139/o59-099

Borlongan, I.G., & Satoh, S. (2001). Dietary phosphorus requirement of juvenile milkfish, Chanos chanos (Forsskal). Aquaculture Research, 32, 26-32. https://doi.org/10.1046/j.1355557x.2001.00003.x

Bureau, D.P., & Cho, C.Y. (1999). Phosphorus utilization by rainbow trout (Oncorhynchus mykiss): estimation of dissolved phosphorus waste output. Aquaculture, 179, 127-140. https://doi.org/10.1016/S0044-8486(99)00157-X

Cara, J.B., Aluru, N., Moyano, F.J., & Vijayan, M.M. (2005). Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 142(4), 426-431. https://doi.org/10.1016/j.cbpb.2005.09.005

Cimino, E.J., Owens, L., Bromage, E., & Anderson, T.A. (2002). A newly developed ELISA showing the effect of environmental stress on levels of hsp86 in Cherax quadricarinatus and Penaeus monodon. Comparative Biochemistry and Physiology, 132A, 591-598. https://doi.org/10.1016/s1095-6433(02)00101-0

Dubeau, S.F., Pan, F., Tremblay, G.C., & Bradley, T.M. (1998). Thermal shock of salmon in vivo induces that heat shock protein hsp70 and confers protection against osmotic shock. Aquaculture, 168, 311-323. https://doi.org/10.1016/S0044-8486(98)00358-5

Ellis, A.E. (1981). Nonspecific defence mechanism in fish and their role in diseases processes. Developments in Biology and Chemistry, 263, 337-352.

Ellis, R.J., van der Vies, S.M., & Hemmingsen, S.M. (1989). The molecular chaperone concept. Biochemistry SOCS. ymp. 55, 145-153.

Ellis, A.E. (1990). Techniques in fish immunology. In: J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Robertson & W.B. Van Muiswinkle, (Eds.). Lysozyme Assayses (pp. 101-103). SOS Publications, Fair Haven.

Ellis, R.J. (1999). The molecular chaperone concept. Cell Biology, 1, 1-9. https://doi.org/10.1098/rstb.1993.0023.

Eya, J.C., & Lovell, R.T. (1998). Effects of dietary phosphorus on resistance of channel catfish to Edwardsiella ictaluri challenge. Journal of Aquatic Animal Health, 10, 28-34. https://doi.org/10.1577/1548-8667(1998)010<0028:EODPOR>2.0.CO;2

Fletcher, T.C. (1982). Non-specific defense mechanism of fish. Developments of Comparative Immunology, 2, 123-132.

Grinde B. (1989). Lysozyme from rainbow trout Salmo gairdneri Richardson as an antibacterial agent against fish pathogens. Journal of Fish Diseases, 12, 207–210.

Ingram, G.A. (1980). Substances involved in the natural resistance of fish to infection- a review. Journal of Fish Biology, 16, 23-60. https://doi.org/10.1111/j.1095-8649.1980.tb03685.x

Iwama, G.K., Thomas, P.T., Forsyth, R.B., & Vijayan, M.M. (1998). Heat shock protein expression in fish. Reviews in Fish Biology, 8, 35-56. https://doi.org/10.1093/icb/39.6.901

Iwama, G.K., Vijayan, M.M., Forsyth, R.B., & Ackerman, P.A. (1999). Heat shock proteins and physiological stress in fish. American Zoology, 39, 901-909. https://doi.org/10.1093/icb/39.6.901

Jokinen, E.I., Vielma, J., Aaltonen, T.M., & Koskela, J. (2003).The effect of dietary phosphorus deficiency on the immune responses of European whitefish (Coregonus lavaretus L.). Fish & Shellfish Immunology, 15, 159-168. https://doi.org/10.1016/S1050-4648(02)00155-9

Kim, J.D., Kim, K.S., Song, J.S., Lee, J.Y., & Jeong, K.S. (1998). Optimum level of dietary monocalcium phosphate based on growth and phosphorus excretion of mirror carp, Cyprinus carpio. Aquaculture, 161, 337-344. https://doi.org/10.1016/S0044-8486(97)00281-0

Kakuta, I., Kurokura, H., Nakamura, H., & Yamauchi, K. (1996). Enhancement of the nonspecific defense activity of the skin mucus of red sea bream by orally administration of bovine lactoferrin. Suisanzoshoku, 44, 197-202. https://doi.org/10.11233/aquaculturesci1953.44.197

Lall, S.P. (2002). The minerals. In: J.E. Halver, R.W. Hardy (Eds.). Fish Nutrition (pp. 260-301). Academic Press, San Diego, California, USA.

Lehninger, A.L., Nelson, D.L., & Coz, M.M. (1993). Principles of Biochemistry. Irving Place, NY: Worth Publishers.

Lewis, S., Handy, R.D., Cordi, B., Billinghurst, Z., & Depledge, M.H. (1999). Stress proteins (HSP’s): methods of detection and their use as an environmental biomarker. Ecotoxicolology 8, 351-368. https://doi.org/10.1023/A:1008982421299

Lovell, T. (1989). Nutrition and Feeding of Fish. AVI book. Van Nostrand Reinhold. 260 pp.

Lowry, O.H., & Lopez, J.A. (1946). The determination of inorganic phosphate in the presence of labile phosphate esters. Journal of Biology and Chemistry, 62, 421-428. https://doi.org/10.1016/s0021-9258(17)41386-x

Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951). Protein measurement with the folin fenol reagent. Journal of Biology and Chemistry, 193, 265-275. https://doi.org/10.1016/S0021-9258(19)52451-6

NRC (National Research, Council), (1993). Nutrient Requirements of Fish. National Academy Press, Washington, DC, USA. 114 pp.

Oliva-Teles, A., & Pimentel-Rodrigues, A. (2004). Phosphorus requirement of European Sea bass (Dicentrarchus labrax L.) juveniles. Aquaculture Research, 35, 636–642. https://doi.org/10.1111/j.1365-2109.2004.01059.x

Olsen, R.E., Hansen, A.C., Rosenlund, G., Hemre, G.I., Mayhew, T.M., Knudsen, D.L., Eroldogan, O.T., Myklebust, R., & Karlsen, O. (2007). Total replacement of fish meal with plant proteins in diets for Atlantic cod (Gadus morhua L.) II – Health aspects. Aquaculture, 272, 612-624. https://doi.org/10.1016/j.aquaculture.2007.05.010

Pillay, T.V.R. (1992). Aquaculture and the Environment. John Wiley and Sons, Inc., New York, NY, USA. 200 pp.

Pimentel-Rodrigues, A.M., & Oliva-Teles, A. (2001). Phosphorus requirement of gilthead sea bream (Sparus aurata L.) juveniles. Aquaculture Research, 32, 157-161. https://doi.org/10.1046/j.1355-557x.2001.00013.x

Ren, T., Koshio, S., Ishikawa, M., Yokoyama, S., Micheal, F.R., Uyan, O., & Tung, H.T. (2007). Influence of dietary vitamin C and bovine lactoferrin on blood chemistry and non-specific immune responses of Japanese eel, Anguilla japonica. Aquaculture, 267, 31-37. https://doi.org/10.1016/j.aquaculture.2007.03.033

Roy, P.K., Lall, S., 2003. Dietary phosphorus requirement of juvenile haddock (Melanogrammus aeglefinus L.). Aquaculture, 221, 451-468. https://doi.org/10.1016/S0044-8486(03)00065-6

Snyder, M.J., Girvets, E., & Mulder, E.P. (2001). Induction of marine mollusc stress proteins by chemical or physical stress. Archieves of Environmental Contamination and Toxicology, 41, 22-29. https://doi.org/10.1007/s002440010217

Storebakken, T., Shearer, K.D., & Roem, A.J. (2000). Growth, uptake and retention of nitrogen and phosphorus, and absorption of other minerals in Atlantic salmon Salmo salar fed diets with fishmeal and soy–protein concentrate as the main sources of protein. Aquaculture Nutrition, 6, 103-108. https://doi.org/10.1046/j.1365-2095.2000.00135.x

Takahashi, Y., Itami, T., Konegawa, K. (1986). Enzymatic properties of partially lysozyme from the skin mucus of carp. Nippon Suisan Gakkaishi, 52, 1209-1214. https://doi.org/10.2331/suisan.52.1209

Tacon, A.G.J. (1990). The essential nutrients. In: A.G.J. Tacon (Eds.). Standard Methods for the Nutrition and Feeding of Farmed Fish and Shrimp (pp. 70-84.), vol. 1. Argent Laboratories Press, Redmond, WA.

Thomson, I., White, A., Fletcher, T.C., Houlihan, D.F., & Secombes, C.J. (1993). The effect of stress on immune response of Atlantic salmon (Salmo salar L.) fed diets containing different amounts of vitamin C. Aquaculture, 114, 1-18. https://doi.org/10.1016/0044-8486(93)90246-U

Uyan, O., Koshio, S., Ishikawa, M., Uyan, S., Ren, T., Yokoyama, S., Komilus, C.F., & Michael, F.R. (2007). Effects of dietary phosphorus and phospholipid level on growth, and phosphorus deficiency signs in juvenile Japanese flounder, Paralichthys olivaceus. Aquaculture, 267, 44-54. https://doi.org/10.1016/j.aquaculture.2007.01.020

Vijayan, M.M., & Moon, T.W. (1992). Acute handling stress alters hepatic glycogen metabolism in food deprived rainbow trout (Oncorhynchus mykiss). Canadian Journal of Fisheries and Aquatic Science, 49, 2260-2266. https://doi.org/10.1139/f92-24

Vijayan, M.M. & Moon, T.W. (1994). The stress response and the plasma disappearance of corticosteroid and glucose in a marine teleost, the sea raven. Canadian Journal of Zoology, 72, 379-386. https://doi.org/10.1139/z94-05

Wang, X., Choi, S., Park, S., Yoo, G., Kim, K., Kang, J., & Bai, S.B. (2005). Optimum dietary phosphorus level of juvenile Japanese flounder Paralichthys olivaceus reared in the recirculating system. Fisheries Science, 71, 168-173. https://doi.org/10.1111/j.1444-2906.2005.00944.x

Yamamoto, A., & Iida, T. (1995). Bactericidal activity of serum of all-female triploid rainbow trout. Fish Pathology, 30, 123-124. https://doi.org/10.3147/jsfp.30.123

Yokoyama, Y., Hashimoto, H., Kubota, S., Kinoshita, M., Toyohara, H., Sakaguchi, M., Tanaka, M., Seikai, T., & Kanamori, M. (1998). cDNA cloning of heat-inducible HSP70, a 70.6 kDa heat shock protein, in Japanese flounder Paralichthys olivaceus. Fisheries Science, 64, 964-968. https://doi.org/10.2331/fishsci.64.964

Yokoyama, S., Koshio, S., Takakura, N., Oshida, K., Ishikawa, M., Gallardo-Cigarroa, F.J., & Teshima, S. (2005). Dietary bovine lactoferrin enhances tolerance to high temperature stress in Japanese flounder Paralichthys olivaceus. Aquaculture, 249, 367-373. https://doi.org/10.1016/j.aquaculture.2005.03.024




How to Cite

UYAN, O., KOSHIO, S., ISHIKAWA, M., & YOKOYAMA, S. (2022). Effect of dietary phosphorus level on defense system parameters and liver HSP70 concentration in juvenile Japanese flounder, Paralichthys olivaceus: Dietary phosphorus effects in Japanese flounder. MARINE REPORTS (MAREP), 1(2), 62–74. https://doi.org/10.5281/zenodo.7393751



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