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Uncovering the Mechanisms of Shrimp Innate Immune Response by RNA Interference

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Abstract

Because of the importance of shrimp in world aquaculture, there is much interest in understanding their immune system in order to improve their resistance to pathogenic microorganisms. An effective tool in studying genes involved in the immune response in shrimp is RNA interference (RNAi). RNAi, first recognized as an antiviral response against RNA viruses, is a cellular mechanism that is triggered by double-stranded RNAs and results in the degradation of homologous genes. In this review, we describe the current studies of genes in shrimp that employed RNAi technology to elucidate or confirm their functions. We also review the potential of RNAi to elicit antiviral response in shrimp.

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References

  • Adachi K, Hirata T, Nagai K, Fujisawa S, Kinoshita M, Sakaguchi M (1999) Purification and characterization of prophenoloxidase from kuruma prawn Penaeus japonicus. Fish Sci 65:919–925

    CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Ai HS, Liao JX, Huang XD, Yin ZX, Weng SP, Zhao ZY, Li SD, Yu XQ, He JG (2009) A novel prophenoloxidase 2 exists in shrimp hemocytes. Dev Comp Immunol 33:59–68

    Article  PubMed  CAS  Google Scholar 

  • Aladaileh S, Rodney P, Nair SV, Raftos DA (2007) Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata). Comp Biochem Physiol B Biochem Mol Biol 148:470–480

    Article  PubMed  Google Scholar 

  • Amparyup P, Kondo H, Hirono I, Aoki T, Tassanakajon A (2008) Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp Penaeus monodon. Mol Immunol 45:1085–1093

    Article  PubMed  CAS  Google Scholar 

  • Amparyup P, Charoensapsri W, Tassanakajon A (2009) Two prophenoloxidases are important for the survival of Vibrio harveyi challenged shrimp Penaeus monodon. Dev Comp Immunol 33:247–256

    Article  PubMed  CAS  Google Scholar 

  • Aspan A, Huang TS, Cerenius L, Soderhall K (1995) cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc Natl Acad Sci USA 92:939–943

    Article  PubMed  CAS  Google Scholar 

  • Assavalapsakul W, Smith DR, Panyim S (2006) Identification and characterization of a Penaeus monodon lymphoid cell-expressed receptor for the yellow head virus. J Virol 80:262–269

    Article  PubMed  CAS  Google Scholar 

  • Bartlett TC, Cuthbertson BJ, Shepard EF, Chapman RW, Gross PS, Warr GW (2002) Crustins, homologues of an 11.5-kDa antibacterial peptide, from two species of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus. Mar Biotechnol (NY) 4:278–293

    Article  CAS  Google Scholar 

  • Bhuiyan NH, Selvaraj G, Wei Y, King J (2008) Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion. J Exp Bot 60(2):509–521

    Article  PubMed  Google Scholar 

  • Boman HG (1995) Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13:61–92

    Article  PubMed  CAS  Google Scholar 

  • Cerenius L, Soderhall K (2004) The prophenoloxidase-activating system in invertebrates. Immunol Rev 198:116–126

    Article  PubMed  CAS  Google Scholar 

  • Cerenius L, Lee BL, Soderhall K (2008) The proPO-system: pros and cons for its role in invertebrate immunity. Trends Immunol 29:263–271

    Article  PubMed  CAS  Google Scholar 

  • Chen MY, Hu KY, Huang CC, Song YL (2005) More than one type of transglutaminase in invertebrates? A second type of transglutaminase is involved in shrimp coagulation. Dev Comp Immunol 29:1003–1016

    Article  PubMed  CAS  Google Scholar 

  • Cruz J, Nieva C, Mane-Padros D, Martin D, Belles X (2008) Nuclear receptor BgFTZ-F1 regulates molting and the timing of ecdysteroid production during nymphal development in the hemimetabolous insect Blattella germanica. Dev Dyn 237:3179–3191

    Article  PubMed  CAS  Google Scholar 

  • Da Silva CCA (2002) Activation of prophenoloxidase and removal of Bacillus subtilis from the hemolymph of Acheta domesticus (L.) (Orthoptera: Gryllidae). Neotrop Entomol 31:487–491

    Article  Google Scholar 

  • De La Vega E, O'leary NA, Shockey JE, Robalino J, Payne C, Browdy CL, Warr GW, Gross PS (2008) Anti-lipopolysaccharide factor in Litopenaeus vannamei (LvALF): a broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection. Mol Immunol 45:1916–1925

    Article  PubMed  Google Scholar 

  • De Lorgeril J, Gueguen Y, Goarant C, Goyard E, Mugnier C, Fievet J, Piquemal D, Bachere E (2008) A relationship between antimicrobial peptide gene expression and capacity of a selected shrimp line to survive a Vibrio infection. Mol Immunol 45:3438–3445

    Article  PubMed  Google Scholar 

  • Dechklar M, Udomkit A, Panyim S (2008) Characterization of argonaute cDNA from Penaeus monodon and implication of its role in RNA interference. Biochem Biophys Res Commun 367:768–774

    Article  PubMed  CAS  Google Scholar 

  • Destoumieux D, Bulet P, Loew D, Van Dorsselaer A, Rodriguez J, Bachere E (1997) Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (Decapoda). J Biol Chem 272:28398–28406

    Article  PubMed  CAS  Google Scholar 

  • Destoumieux-Garzon D, Saulnier D, Garnier J, Jouffrey C, Bulet P, Bachere E (2001) Crustacean immunity. Antifungal peptides are generated from the C terminus of shrimp hemocyanin in response to microbial challenge. J Biol Chem 276:47070–47077

    Article  PubMed  CAS  Google Scholar 

  • Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200

    Article  PubMed  CAS  Google Scholar 

  • Fagutao FF, Koyama T, Kaizu A, Saito-Taki T, Kondo H, Aoki T, Hirono I (2009) Increased bacterial load in shrimp hemolymph in the absence of prophenoloxidase. FEBS J 276:5298–5306

    Article  PubMed  CAS  Google Scholar 

  • Fire A, Albertson D, Harrison SW, Moerman DG (1991) Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle. Development 113:503–514

    PubMed  CAS  Google Scholar 

  • Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811

    Article  PubMed  CAS  Google Scholar 

  • Golding MC, Long CR, Carmell MA, Hannon GJ, Westhusin ME (2006) Suppression of prion protein in livestock by RNA interference. Proc Natl Acad Sci USA 103:5285–5290

    Article  PubMed  CAS  Google Scholar 

  • Guo S, Kemphues KJ (1995) par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81:611–620

    Article  PubMed  CAS  Google Scholar 

  • Hall M, Wang R, Van Antwerpen R, Sottrup-Jensen L, Soderhall K (1999) The crayfish plasma clotting protein: a vitellogenin-related protein responsible for clot formation in crustacean blood. Proc Natl Acad Sci USA 96:1965–1970

    Article  PubMed  CAS  Google Scholar 

  • Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296

    Article  PubMed  CAS  Google Scholar 

  • Hannon GJ (2002) RNA interference. Nature 418:244–251

    Article  PubMed  CAS  Google Scholar 

  • Huang CC, Sritunyalucksana K, Soderhall K, Song YL (2004) Molecular cloning and characterization of tiger shrimp (Penaeus monodon) transglutaminase. Dev Comp Immunol 28:279–294

    Article  PubMed  CAS  Google Scholar 

  • Hui JH, Tobe SS, Chan SM (2008) Characterization of the putative farnesoic acid O-methyltransferase (LvFAMeT) cDNA from white shrimp, Litopenaeus vannamei: Evidence for its role in molting. Peptides 29:252–260

    Article  PubMed  CAS  Google Scholar 

  • Iwanaga S (2002) The molecular basis of innate immunity in the horseshoe crab. Curr Opin Immunol 14:87–95

    Article  PubMed  CAS  Google Scholar 

  • Iwanaga S, Lee BL (2005) Recent advances in the innate immunity of invertebrate animals. J Biochem Mol Biol 38:128–150

    Article  PubMed  CAS  Google Scholar 

  • Jorgensen R (1990) Altered gene expression in plants due to trans interactions between homologous genes. Trends Biotechnol 8:340–344

    Article  PubMed  CAS  Google Scholar 

  • Kim CS, Kosuke Z, Nam YK, Kim SK, Kim KH (2007) Protection of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV) challenge by double-stranded RNA. Fish Shellfish Immunol 23:242–246

    Article  PubMed  CAS  Google Scholar 

  • Kopacek P, Hall M, Soderhall K (1993) Characterization of a clotting protein, isolated from plasma of the freshwater crayfish Pacifastacus leniusculus. Eur J Biochem 213:591–597

    Article  PubMed  CAS  Google Scholar 

  • Lam PB, Burga LN, Wu BP, Hofstatter EW, Lu KP, Wulf GM (2008) Prolyl isomerase Pin1 is highly expressed in Her2-positive breast cancer and regulates erbB2 protein stability. Mol Cancer 7:91

    Article  PubMed  Google Scholar 

  • Lee S, Söderhäll K (2002) Early events in crustacean innate immunity. Fish Shellfish Immunol 12:421–437

    Article  PubMed  CAS  Google Scholar 

  • Li DF, Zhang MC, Yang HJ, Zhu YB, Xu X (2007) Beta-integrin mediates WSSV infection. Virology 368:122–132

    Article  PubMed  CAS  Google Scholar 

  • Li F, Hui Y, Wang D, Jose Priya TA, Li S, Wang B, Zhang J, Xiang J (2009) Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides. Dev Comp Immunol 33(10):1093–1101

    Google Scholar 

  • Liu F, Liu Y, Li F, Dong B, Xiang J (2005) Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis). Mar Biotechnol (NY) 7:600–608

    Article  CAS  Google Scholar 

  • Lugo JM, Morera Y, Rodriguez T, Huberman A, Ramos L, Estrada MP (2006) Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti. Functional analysis by double-stranded RNA interference technique. Febs J 273:5669–5677

    Article  PubMed  CAS  Google Scholar 

  • Maningas MB, Kondo H, Hirono I, Saito-Taki T, Aoki T (2008) Essential function of transglutaminase and clotting protein in shrimp immunity. Mol Immunol 45:1269–1275

    Article  PubMed  CAS  Google Scholar 

  • Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T (2002) Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563–574

    Article  PubMed  CAS  Google Scholar 

  • Mcgregor AP, Pechmann M, Schwager EE, Feitosa NM, Kruck S, Aranda M, Damen WG (2008) Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider. Curr Biol 18:1619–1623

    Article  PubMed  CAS  Google Scholar 

  • Mori C, Takanami T, Higashitani A (2008) Maintenance of mitochondrial DNA by the Caenorhabditis elegans ATR checkpoint protein ATL-1. Genetics 180:681–686

    Article  PubMed  CAS  Google Scholar 

  • Ongvarrasopone C, Chanasakulniyom M, Sritunyalucksana K, Panyim S (2008) Suppression of PmRab7 by dsRNA inhibits WSSV or YHV infection in shrimp. Mar Biotechnol 10:374–381

    Article  PubMed  CAS  Google Scholar 

  • Palakodeti D, Smielewska M, Lu YC, Yeo GW, Graveley BR (2008) The PIWI proteins SMEDWI-2 and SMEDWI-3 are required for stem cell function and piRNA expression in planarians. RNA 14:1174–1186

    Article  PubMed  CAS  Google Scholar 

  • Patat SA, Carnegie RB, Kingsbury C, Gross PS, Chapman R, Schey KL (2004) Antimicrobial activity of histones from hemocytes of the Pacific white shrimp. Eur J Biochem 271:4825–4833

    Article  PubMed  CAS  Google Scholar 

  • Perazzolo LM, Lorenzini DM, Daffre S, Barracco MA (2005) Purification and partial characterization of the plasma clotting protein from the pink shrimp Farfantepenaeus paulensis. Comp Biochem Physiol B Biochem Mol Biol 142:302–307

    Article  PubMed  Google Scholar 

  • Relf JM, Chisholm JR, Kemp GD, Smith VJ (1999) Purification and characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas. Eur J Biochem 264:350–357

    Article  PubMed  CAS  Google Scholar 

  • Reyes-Izquierdo T, Vargas-Albores F (2001) Proteinase activity in the white shrimp (Penaeus vannamei) clotting protein. Biochem Biophys Res Commun 287:332–336

    Article  PubMed  CAS  Google Scholar 

  • Rijiravanich A, Browdy CL, Withyachumnarnkul B (2008) Knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei challenged with a low dose of white-spot syndrome virus. Fish Shellfish Immunol 24:308–313

    Article  PubMed  CAS  Google Scholar 

  • Robalino J, Browdy CL, Prior S, Metz A, Parnell P, Gross P, Warr G (2004) Induction of antiviral immunity by double-stranded RNA in a marine invertebrate. J Virol 78:10442–10448

    Article  PubMed  CAS  Google Scholar 

  • Sappl PG, Carroll AJ, Clifton R, Lister R, Whelan J, Harvey Millar A, Singh K (2008) The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing multiple genes results in altered metabolic sensitivity to oxidative stress. Plant J, 58(1):53–68

    Google Scholar 

  • Sarathi M, Simon MC, Ahmed VP, Kumar SR, Hameed AS (2008a) Silencing VP28 gene of white spot syndrome virus of shrimp by bacterially expressed dsRNA. Mar Biotechnol (NY) 10:198–206

    Article  CAS  Google Scholar 

  • Sarathi M, Simon MC, Venkatesan C, Hameed AS (2008b) Oral administration of bacterially expressed VP28dsRNA to protect Penaeus monodon from white spot syndrome virus. Mar Biotechnol (NY) 10:242–249

    Article  CAS  Google Scholar 

  • Shockey JE, O'leary NA, De La Vega E, Browdy CL, Baatz JE, Gross PS (2008) The role of crustins in Litopenaeus vannamei in response to infection with shrimp pathogens: An in vivo approach. Dev Comp Immunol 33(5):668–673

    Google Scholar 

  • Soderhall K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10:23–28

    Article  PubMed  CAS  Google Scholar 

  • Somboonwiwat K, Marcos M, Tassanakajon A, Klinbunga S, Aumelas A, Romestand B, Gueguen Y, Boze H, Moulin G, Bachere E (2005) Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon. Dev Comp Immunol 29:841–851

    Article  PubMed  CAS  Google Scholar 

  • Su J, Oanh DT, Lyons RE, Leeton L, Van Hulten MC, Tan SH, Song L, Rajendran KV, Walker PJ (2008) A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1. Fish Shellfish Immunol 24:223–233

    Article  PubMed  Google Scholar 

  • Sun Y, Liu M, Yang B, Lu J, Li B (2008) Inhibition of laryngeal cancer cell invasion and growth with lentiviral-vector delivered short hairpin RNA targeting human mmp-9 gene. Cancer Invest 26:984–989

    Article  PubMed  CAS  Google Scholar 

  • Supungul P, Klinbunga S, Pichyangkura R, Hirono I, Aoki T, Tassanakajon A (2004) Antimicrobial peptides discovered in the black tiger shrimp Penaeus monodon using the EST approach. Dis Aquat Org 61:123–135

    Article  PubMed  CAS  Google Scholar 

  • Tanaka S, Nakamura T, Morita T, Iwanaga S (1982) Limulus anti-LPS factor: an anticoagulant which inhibits the endotoxin mediated activation of Limulus coagulation system. Biochem Biophys Res Commun 105:717–723

    Article  PubMed  CAS  Google Scholar 

  • Tharntada S, Somboonwiwat K, Rimphanitchayakit V, Tassanakajon A (2008) Anti-lipopolysaccharide factors from the black tiger shrimp, Penaeus monodon, are encoded by two genomic loci. Fish Shellfish Immunol 24:46–54

    Article  PubMed  CAS  Google Scholar 

  • Tincu JA, Taylor SW (2004) Antimicrobial peptides from marine invertebrates. Antimicrob Agents Chemother 48:3645–3654

    Article  PubMed  CAS  Google Scholar 

  • Tirasophon W, Roshorm Y, Panyim S (2005) Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA. Biochem Biophys Res Commun 334:102–107

    Article  PubMed  CAS  Google Scholar 

  • Tirasophon W, Yodmuang S, Chinnirunvong W, Plongthongkum N, Panyim S (2007) Therapeutic inhibition of yellow head virus multiplication in infected shrimps by YHV-protease dsRNA. Antiviral Res 74:150–155

    Article  PubMed  CAS  Google Scholar 

  • Tiu SH-K, Chan S (2007) The use of recombinant protein and RNA interference approaches to study the reproductive functions of a gonad-stimulating hormone from the shrimp Metapenaeus ensis. FEBS J 274:4385–4395

    Article  PubMed  CAS  Google Scholar 

  • Tiu SH, He JG, Chan SM (2007) The LvCHH-ITP gene of the shrimp (Litopenaeus vannamei) produces a widely expressed putative ion transport peptide (LvITP) for osmo-regulation. Gene 396:226–235

    Article  PubMed  CAS  Google Scholar 

  • Tiu SH, Benzie J, Chan SM (2008) From hepatopancreas to ovary: molecular characterization of a shrimp vitellogenin receptor involved in the processing of vitellogenin. Biol Reprod 79:66–74

    Article  PubMed  CAS  Google Scholar 

  • Treerattrakool S, Panyim S, Chan SM, Withyachumnarnkul B, Udomkit A (2008) Molecular characterization of gonad-inhibiting hormone of Penaeus monodon and elucidation of its inhibitory role in vitellogenin expression by RNA interference. FEBS J 275:970–980

    Article  PubMed  CAS  Google Scholar 

  • Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13:3191–3197

    Article  PubMed  CAS  Google Scholar 

  • Vaucheret H, Beclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114:3083–3091

    PubMed  CAS  Google Scholar 

  • Wang R, Liang Z, Hal M, Soderhall K (2001) A transglutaminase involved in the coagulation system of the freshwater crayfish. Pacifastacus leniusculus. Tissue localisation and cDNA cloning. Fish Shellfish Immunol 11:623–637

    Article  PubMed  CAS  Google Scholar 

  • Wang L, Zhi B, Wu W, Zhang X (2008) Requirement for shrimp caspase in apoptosis against virus infection. Dev Comp Immunol 32:706–715

    Article  PubMed  CAS  Google Scholar 

  • Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defence against viruses. Nature 411:834–842

    Article  PubMed  CAS  Google Scholar 

  • Westenberg M, Heinhuis B, Zuidema D, Vlak JM (2005) siRNA injection induces sequence-independent protection in Penaeus monodon against white spot syndrome virus. Virus Res 114:133–139

    Article  PubMed  CAS  Google Scholar 

  • Wu Y, Lü L, Yang L-S, Weng S-P, Chan S-M, He J-G (2007) Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequence-specific siRNA. Aquaculture 271:21–30

    Article  CAS  Google Scholar 

  • Wu W, Zong R, Xu J, Zhang X (2008) Antiviral phagocytosis is regulated by a novel Rab-dependent complex in shrimp Penaeus japonicus. J Proteome Res 7:424–431

    Article  PubMed  CAS  Google Scholar 

  • Xu J, Han F, Zhang X (2007) Silencing shrimp white spot syndrome virus (WSSV) genes by siRNA. Antiviral Res 73:126–131

    Article  PubMed  CAS  Google Scholar 

  • Xu J, Wu S, Zhang X (2008) Novel function of QM protein of shrimp (Penaeus japonicus) in regulation of phenol oxidase activity by interaction with hemocyanin. Cell Physiol Biochem 21:473–480

    Article  PubMed  CAS  Google Scholar 

  • Yeh MS, Huang CJ, Leu JH, Lee YC, Tsai IH (1999) Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon). Eur J Biochem 266:624–633

    Article  PubMed  CAS  Google Scholar 

  • Yodmuang S, Tirasophon W, Roshorm Y, Chinnirunvong W, Panyim S (2006) YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality. Biochem Biophys Res Commun 341:351–356

    Article  PubMed  CAS  Google Scholar 

  • Zasloff M (1992) Antibiotic peptides as mediators of innate immunity. Curr Opin Immunol 4:3–7

    Article  PubMed  CAS  Google Scholar 

  • Zong R, Wu W, Xu J, Zhang X (2008) Regulation of phagocytosis against bacterium by Rab GTPase in shrimp Marsupenaeus japonicus. Fish Shellfish Immunol 25:258–263

    Article  PubMed  CAS  Google Scholar 

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Hirono, I., Fagutao, F.F., Kondo, H. et al. Uncovering the Mechanisms of Shrimp Innate Immune Response by RNA Interference. Mar Biotechnol 13, 622–628 (2011). https://doi.org/10.1007/s10126-010-9292-0

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