Effect of microbial immunostimulants on WSSV infection percentage and the expression of immune-related genes in white shrimp (Litopenaeus vannamei)

  • Jesús A. Fierro Coronado National Polytechnic Institute, Autonomous University of Baja California
  • Antonio Luna González National Polytechnic Institute
  • Carlos J. Caceres Martínez Autonomous University of Baja California Sur
  • Píndaro Álvarez Ruiz National Polytechnic Institute
  • Ruth Escamilla-Montes National Polytechnic Institute
  • Héctor A. González Ocampo National Polytechnic Institute
  • Viridiana Peraza-Gómez Autonomous University of Nayarit
Keywords: Candida parapsilosis, gene expression, immune system, Litopenaeus vannamei, Pediococcus parvulus, shrimp, white shrimp


Background: The white spot syndrome virus (WSSV) causes high mortalities in aquaculture. The use of immunostimulants increases animal resistance. Objective: To evaluate the WSSV infection percentage and the immunostimulant effect of lactic acid bacteria and yeast (MI= microbial immunostimulants) on WSSV infected Litopenaeus vannamei. Methods: A bioassay was performed for 33 d, with treatments in triplicate. The MI was added to the feed at 8.5 mg/kg feed and offered to shrimp (9.9 ± 3.1 g) daily, every 2 days, or every 3 days. Shrimp were infected with WSSV at 9 and 19 days. The expression of four immune system-related genes was studied using qRT-PCR. Results: No significant differences were observed in growth and survival among treatments. At the end of the bioassay, WSSV infection percentage (low viral load) decreased 8.3 and 25% in treatments III and IV as compared to the control group. Treatments with MI showed significant differences in the relative expression of LvToll, transglutaminase, and prophenoloxidase genes when MI was offered daily as compared to the control group. The MI did not regulate the expression of the superoxide dismutase gene. The WSSV infection percentage decreased when feed with MI was offered every 3 d. Conclusion: The MI decrease WSSV infection percentage in L. vannamei infected with low viral load when it is offered every three days. The MI up-regulates LvToll, Tgase, and proPO genes when it is offered daily. Further research is needed regarding prophylactic treatment with microbial immunostimulants against WSSV in commercial shrimp farms.

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Author Biographies

Jesús A. Fierro Coronado, National Polytechnic Institute, Autonomous University of Baja California

National Polytechnic Institute, Interdisciplinary Research Center for Integral Regional Development, Sinaloa Unit. Aquaculture Department, Guasave, Sinaloa, Mexico. Autonomous University of Baja California Sur, La Paz, México.

Antonio Luna González, National Polytechnic Institute

National Polytechnic Institute, Interdisciplinary Research Center for Integral Regional Development, Sinaloa Unit. Aquaculture Department, Guasave, Sinaloa, México.

Carlos J. Caceres Martínez, Autonomous University of Baja California Sur

Autonomous University of Baja California Sur, La Paz, México.

Píndaro Álvarez Ruiz, National Polytechnic Institute

National Polytechnic Institute, Interdisciplinary Research Center for Integral Regional Development, Sinaloa Unit. Aquaculture Department, Guasave, Sinaloa, México.

Ruth Escamilla-Montes, National Polytechnic Institute

National Polytechnic Institute, Interdisciplinary Research Center for Integral Regional Development, Sinaloa Unit. Aquaculture Department, Guasave, Sinaloa, México.

Héctor A. González Ocampo, National Polytechnic Institute

National Polytechnic Institute, Interdisciplinary Research Center for Integral Regional Development, Sinaloa Unit. Aquaculture Department, Guasave, Sinaloa, México.

Viridiana Peraza-Gómez, Autonomous University of Nayarit

Autonomous University of Nayarit, Amado Nervo City of Culture, Tepic, Nayarit, México.


Ai HS, Huang YC, Li SD, Weng SP, Yu XQ, He JG. Characterization of a prophenoloxidase from hemocytes of the shrimp Litopenaeus vannamei that is down-regulated by white spot syndrome virus. Fish Shellfish Immunol 2008; 25: 28-39.

Akira S, Hemmi H. Recognition of pathogen-associated molecular pattern by TLR family. Immunol Lett 2003; 85: 85–95.

Píndaro Alvarez-Ruiz, Antonio Luna-González, Ruth Escamilla-Montes, Claudio H. Mejía-Ruiz and Francisco J. Magallón-Barajas, Raúl Llera-Herrera, Diego A. Galván-Alvarez. Long-lasting effect against white spot syndrome virus in shrimp broodstock, Litopenaeus vannamei, by LvRab7 Silencing. J World Aquacult Soc 2015; 46(6): 571-582.

Andersen CL, Jensen JL, Orntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004; 64(15): 5245–5250.

Andrews SR, Sahu N.P, Pal AK, Mukherjee SC, Kumar S. Yeast extract, brewer’s yeast and spirulina in diets for Labeo rohita fingerlings affect haemato-immunological responses and survival following Aeromonas hydrophila challenge. Res Vet Sci 2011; 91: 103-109.

Amparyup P, Charoensapsri W, Tassanakajon A. Prophenoloxidase system and its role in shrimp immune responses against major pathogens. Fish Shellfish Immunol 2013; 4: 990-1001.

Apún-Molina JP, Santamaría Miranda A, Luna González A, Martínez Díaz S F, Rojas Contreras M. Effect of potential probiotic bacteria on growth and survival of tilapia Oreochromis niloticus L., cultured in the laboratory under high density and suboptimumtemperature. Aquac Res2009; 40: 887–894.

Arts JAJ, Cornelissen FHJ, Cijsouw T, Hermsen T, Savelkoul HFJ, Stet RJM. Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon. Fish shellfish Immunol2007; 23: 504-513.

Babu DT, Swapna PA, Simi PJ, Bright AR, Philip R. Marine yeast Candida aquaetextoris S527 as a potential immunostimulant in black tiger shrimp Penaeus monodon. J Invertebr Pathol 2013; 112: 243–252.

Bai N, Zhang W, Mai K, Wang X, Xu W. Ma H. Effects of discontinuous administration of β-glucan and glycyrrhizin on the growth and immunity of white shrimp Litopenaeus vannamei. Aquaculture 2010; 306: 218-224.

Boyd CE, Tucker CS. Pond Aquaculture Water Quality Management. Boston (EE.UU): Kluwer Academic Publishers; 1988.

Brock AJ, Main KL. A guide to the Common Problems and Disease of Cultured Penaeus vannamei. 1st Edn. The Oceanic Institute 1994; Honolulu-Hi, ISBN: 1-886608-00-8, 90-94.

Campa-Córdova AI, Hernández Saavedra NY, Aguirre Guzmán G, Ascencio F. Immunomodulatory response of superoxide dismutase in juvenile American white shrimp (Litopenaeus vannamei) exposed to immunostimulants. Cienc Mar2005; 31(4): 661–669.

Cerenius L, Söderhäll K. The prophenoloxidase-activating system in invertebrates. Immunol Rev 2004;198; 116-126.

Cheng G, Hao H, Xie S, Wang X, Dai M, Huang L, Yuan Z. Antibiotic alternatives: the substitution of antibiotics in animal husbandry? Front Microbiol 2014; 5, 217. doi: 10.3389/fmicb.2014.00217.

Destoumieux D, Muñoz M, Cosseau C, Rodríguez J, Bulet P. Comps M, Bachère E. Penaedins, antimicrobial peptides with chitin binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge. J Cell Sci2000; 113: 461-469.

FAO. The State of world fisheries and aquaculture. FAO Fisheries and Aquaculture Department. Food and Agriculture Organization of the United Nations. 2016. 230 pp.

Flores-Miranda MC, Luna González A, Campa Córdova AI, González Ocampo HA, Fierro Coronado JA, Partida Arangure BO. Microbial immunostimulants reduce mortality in whiteleg shrimp (Litopenaeus vannamei) challenged with Vibrio sinaloensis strains. Aquaculture 2011; 320: 51–55.

Gatlin DM III. Nutrition and fish health. In: Fish nutrition (ed. by J.E. Halver and R. W. Hardy): San Diego, (CA), USA Academic press; 2002: 671-702 p.

Goncalves P, Guertler C, Bachère E, de Souza CRB, Rosa RD, Perazzolo LM. Molecular signatures at imminent death: Hemocyte gene expression profiling of shrimp succumbing to viral and fungal infections. Dev Comp Immunol 2014; 42: 294–301.

Itami I, Asano M, Tokushige K, Kubono K, Nakagawa A, Takeno N, Nishimura H, Maeda M, Kondo M, Takahashi Y. Enhancement of disease resistance of kuruma shrimp, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum. Aquaculture 1998; 164: 277-288.

Jiravanichpaisal P, Lee BL, Söderhäll K. Cell-mediated immunity in arthropods: Hematopoiesis, coagulation, melanization and opsonization. Immunobiology2006; 11: 213-236.

Joseph TC, James R, Rajan LA, Surendran PK, Lalitha K. Occurrence of viral pathogens in Penaeus monodon post-larvae from aquaculture hatcheries. Data Brief 2015; 4: 170–176.

Kimura T, Yamano K, Nakano H, Momoyama K, Hiraoka M, Inouye K. Detection of penaeid-rod shaped DNA virus (PRDV) by PCR. Fish Pathol 1996; 31(2): 93-98.

Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu Rev Immunol 2007;25: 697–743.

Lightner DV. Virus diseases of farmed shrimp in the Western Hemisphere (the Americas): A review J Invertebr Pathol 2011; 106: 110-130.

Liu YC, Li FH, Wang B, Dong B, Zhang QL, Luan W. A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning, tissue localization and expression profile after challenge. Fish Shellfish Immunol 2007; 22(5): 576-588.

Lo CF, Leu JH, Ho CH, Chen CH, Peng SE, Chen YT, Chou CM, Yeh P Y, Huang CJ, Chou HY, Wang CH, Kou GH. Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Dis Aquat Org 1996a; 25: 133–141.

Lo CF, Ho CH, Peng SE, Chen CH, Hsu HC, Chiu YL, Chang CF, Liu K.F, Su MS, Wang CH, Kou GH. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis Aquat Org 1996b; 27: 215–225.

Luna-González, A., Moreno-Herrera, J.T., Campa-Córdova, A.I., González-Ocampo, H.A, Fierro-Coronado, J.A., Álvarez-Ruiz, P., Bueno-Ibarra, M.A. Respuesta inmune y expresión de genes en el camarón blanco (Litopenaeus vannamei) inducida por inmunoestimulantes microbianos. Lat Am J Aquat Res 2013a; 41(5): 898-907.

Luna-González A, Quiñónez Zúñiga D, Fierro Coronado JA, González Ocampo HA, Campa Córdova AI, Flores Miranda MC, Peraza Gómez, V. Effect of Pediococcus parvulus and Candida parapsilosis on growth and survival of tilapia. Oreochromis niloticusand Oreochromis sp. Afr J Microb Res 2013b; 7(23): 2976–2982.

McPherson MJ, Hames BD, Taylor GR. PCR a Practical Aproach. First Editon edn. Oxford University Press Oxford; 2008.

Montaño-Pérez K, Reyes Izquierdo T, Vargas Albores F. El proceso de coagulación en camarones penaeidos. Ciencia 1999; 50: 23-28.

Morales V, Cuéllar A. Guía Técnica – Patología e Inmunología de Camarones Penaeidos. eds. Panamá (República de Panamá); 2014; p. 237-308.Oduleye SO. Growth and growth regulation in the cichlids. Aquaculture 1982; 27: 301-306.

Partida-Arangure BO, Luna González A, Fierro Coronado JA, Flores Miranda MC, González Ocampo HA. Effect of inulin and probiotic bacteria on growth, survival, immune response, and prevalence of white spot syndrome virus (WSSV) in Litopenaeus vannamei cultured under laboratory conditions. Afr J Biotechnol 2013; 12(21): 3366-3375.

Ringø E, Olsen RE, GonzálezVecino JL, Wadsworth S, Song SK. Use of immunostimulants and nucleotides in aquaculture: a review. J Marine Sci. Res Development 2012; 1, 104. doi:10.4172/2155-9910.1000104.

Sajeevan TP, Rosamma P, Bright, Singh. I.S. Dose/frequency: a critical factor in the administration of glucan as immunostimulant to Indian white shrimp, Fenneropenaeus indicus. Aquaculture2009a; 287: 248–252.

Sajeevan TP, Lowman DW, Williams DL, Selven S, Anas A, Rosamma, P. Marine yeast diet confers better protection than its cell wall component (1–3)-β-D-glucan as an immunostimulant in Fenneropenaeus indicus. Aquacult Res 2009b; 40: 1723–1730.

Song Y., Hsieh YT. Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for generation of microbiocidal substances: analysis of reactive oxygen species. Dev Comp Immunol 1994; 18: 201–209.

Stephens A, Rojo L, Araujo-Bernal S, García-Carreño F, Muhlia-Almazán A. Cathepsin B from the white shrimp Litopenaeus vannamei: cDNA sequence analysis, tissues-specific expression and biological activity. Comp Biochem Physiol B 2012; 161: 32–40.

Strickland JDH, Parsons TR. A practical handbook of seawater analysis. Bullin Fisheries Research Board of Canada 1972; 167: 310.

Sun J, Wang A, Zhang T. Flow cytometric analysis of defense functions of hemocytes from the penaeid shrimp, Penaeus vannamei. J World Aquac Soc 2010; 41(1): 92–105.

Tang K, Lightner DV. Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR. Dis Aquat Organ 2001; 44: 79–85.

Vandesompele J, Preter KD, Pattyn F, Poppe B, Roy NV, Paepe AD, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002. 3(7), research0034.1

Vargas-Albores F, Guzmán M, Ochoa J. An anticoagulant solution for haemolymph collectionand prophenoloxidase studies of penaeid shrimp (Penaeus californiensis). Comp Biochem Physiol 1993; 106: 299-303.

Wang YC, Chang PS, Chen HY. Tissue expressions of nine genes important to immune defence of the Pacific white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 2007; 23: 1161-1177.

Wang KCHC, Tseng CW, Lin HY, Chen IT, Chen YH, Chen YM, Chen YM, Chen TY, Yang HL. RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi. Dev Comp Immunol2010;34: 49–58.

Wilsi W, Douglas L, Swapna PA, Jayesh P, J, IS Bright, Rosamma. Immune gene expression profile of Penaeus monodon in response to marine yeast glucan application and white spot syndrome virus challenge. Fish Shellfish Immunol 2015; 43: 346-356.

Yeh SP, Chen YN, Hsieh SL, Cheng W, Liu CH. Immune response of white shrimp, Litopenaeus vannamei, after a concurrent infection with white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus. Fish Shellfish Immunol2009; 26: 582–588.

Zar JH. Biostatistical analysis. 3rd ed. Englewood (Cliffs): Prentice-Hall; 1996. 662 pp.

How to Cite
Fierro Coronado J. A., Luna González A., Caceres Martínez C. J., Álvarez Ruiz P., Escamilla-Montes R., González Ocampo H. A., & Peraza-Gómez V. (2019). Effect of microbial immunostimulants on WSSV infection percentage and the expression of immune-related genes in white shrimp (Litopenaeus vannamei). Revista Colombiana De Ciencias Pecuarias, 32(3), 221-231. https://doi.org/10.17533/udea.rccp.v32n3a07
Original research articles