Beetle Immunity

  • Ji-Won Park
  • Chan-Hee Kim
  • Jiang Rui
  • Keun-HwaPark
  • Kyung-Hwa Ryu
  • Jun-Ho Chai
  • Hyun-Ok Hwang
  • Kenji Kurokawa
  • Nam-Chul Ha
  • Irene Söderhäll
  • Kenneth Söderhäll
  • Bok Luel Lee
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 708)


Genetic studies have elegantly characterized the innate immune response in Drosophila melanogaster. However, these studies have a limited ability to reveal the biochemical mechanisms underlying the innate immune response. To investigate the biochemical basis of how insects recognize invading microbes and how these recognition signals activate the innate immune response, it is necessary to use insects, from which larger amounts of hemolymph can be extracted. Using the larvae from two species of beetle, Tenebrio molitor and Holotrichia diomphalia, we elucidated the mechanisms underlying pathogenic microbe recognition. In addition, we studied the mechanism of host defense molecule amplification. In particular, we identified several pattern recognition proteins, serine proteases, serpins and antimicrobial peptides and examined how these molecules affect innate immunity.


Coleopteran Insect Clip Domain Stal Structure Holotrichia Diomphalia 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    Sokoloff A. The Biology of Tribolium with Special Emphasis on Genetic Aspects. Oxford: Oxford Universty Press, 1974.Google Scholar
  2. 2.
    Richards S, Gibbs RA, Weinstock GM et al. The genome of the model beetle and pest Tribolium castaneum. Nature 2008; 452:949–955.PubMedCrossRefGoogle Scholar
  3. 3.
    Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006; 124:783–801.PubMedCrossRefGoogle Scholar
  4. 4.
    Medzhitov R, Janeway CA, Jr. Innate immunity: the virtues of a nonclonal system of recognition. Cell 1997; 91:295–98.PubMedCrossRefGoogle Scholar
  5. 5.
    Medzhitov R, Janeway CA, Jr. Decoding the patterns of self and nonself by the innate immune system. Science 2002; 296:298–300.PubMedCrossRefGoogle Scholar
  6. 6.
    Hoffmann JA, Kafatos FC, Janeway CA et al. Phylogenetic perspectives in innate immunity. Science 1999; 284:1313–1318.PubMedCrossRefGoogle Scholar
  7. 7.
    Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol 2000; 12:13–19.PubMedCrossRefGoogle Scholar
  8. 8.
    Royet J, Reichhart JM, Hoffmann JA. Sensing and signaling during infection in Drosophila. Curr Opin Immunol 2005; 17:11–17.PubMedCrossRefGoogle Scholar
  9. 9.
    Cherry S, Silverman N. Host-pathogen interactions in drosophila: new tricks from an old friend. Nat Immunol 2006; 7:911–917.PubMedCrossRefGoogle Scholar
  10. 10.
    Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu Rev Immunol 2007; 25:697–743.PubMedCrossRefGoogle Scholar
  11. 11.
    Michel T, Reichhart JM, Hoffmann J et al. Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature 2001; 414:756–759.PubMedCrossRefGoogle Scholar
  12. 12.
    Leulier F, Parquet C, Pili-Floury S et al. The Drosophila immune system detects bacteria through specific peptidoglycan recognition. Nat Immunol 2003; 4:478–484.PubMedCrossRefGoogle Scholar
  13. 13.
    Park JW, Kim CH, Kim JH et al. Clustering of peptidoglycan recognition protein-SA is required for sensing lysine-type peptidoglycan in insects. Proc Natl Acad Sci USA 2007; 104:6602–6607.PubMedCrossRefGoogle Scholar
  14. 14.
    Levashina EA, Langley E, Green C et al. Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. Science 1999; 285:1917–1919.PubMedCrossRefGoogle Scholar
  15. 15.
    Gottar M, Gobert V, Matskevich AA et al. Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 2006; 127:1425–1437.PubMedCrossRefGoogle Scholar
  16. 16.
    Holmskov U, Thiel S, Jensenius JC. Collections and ficolins: humoral lectins ofthe innate immune defense. Annu Rev Immunol 2003; 21:547–578.PubMedCrossRefGoogle Scholar
  17. 17.
    Gillespie JP, Kanost MR, Trenczek T. Biological mediators of insect immunity. Annu Rev Entomol 1997; 42:611–643.PubMedCrossRefGoogle Scholar
  18. 18.
    Cerenius L, Lee BL, Söderhäll K. The proPO-system: pros and cons for its role in invertebrate immunity. Trends Immunol 2008; 29:263–271.PubMedCrossRefGoogle Scholar
  19. 19.
    Lee SY, Cho MY, Hyun JH et al. Molecular cloning of cDNA for pro-phenol-oxidase-activating factor I, a serine protease is induced by lipopolysaccharide or 1,3-beta-glucan in coleopteran insect, Holotrichia diomphalia larvae. Eur J Biochem 1998; 257:615–621.PubMedCrossRefGoogle Scholar
  20. 20.
    Jiang H, Wang Y, Kanost MR. Pro-phenol oxidase activating proteinase from an insect, Manducasexta: a bacteria-inducible protein similar to Drosophila easter. Proc Natl Acad Sci USA 1998; 95:12220–12225.PubMedCrossRefGoogle Scholar
  21. 21.
    Satoh D, Horii A, Ochiai M et al. Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization and cDNA cloning. J Biol Chem 1999; 274:7441–7453.PubMedCrossRefGoogle Scholar
  22. 22.
    Wang R, Lee SY, Cerenius L et al. Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus. Eur J Biochem 2001; 268:895–902.PubMedCrossRefGoogle Scholar
  23. 23.
    Sugumaran M. Molecular mechanisms for mammalian melanogenesis. Comparison with insect cuticular sclerotization. FEBS Lett 1991; 295:233–239.PubMedCrossRefGoogle Scholar
  24. 24.
    Lee SY, Moon HJ, Kurata S et al. Purification and molecular cloning of cDNA for an inducible antibacterial protein of larvae of a coleopteran insect, Holotrichia diomphalia. J Biochem 1994; 115:82–86.PubMedGoogle Scholar
  25. 25.
    Moon HJ, Lee SY, Kurata S et al. Purification and molecular cloning of cDNA for an inducible antibacterial protein from larvae of the coleopteran, Tenebrio molitor. J Biochem 1994; 116:53–58.PubMedGoogle Scholar
  26. 26.
    Lee SY, Moon HJ, Kawabata S et al. A sapecin homologue of Holotrichia diomphalia: purification, sequencing and determination of disulfide pairs. Biol Pharm Bull 1995; 18:457–459.PubMedCrossRefGoogle Scholar
  27. 27.
    Lee SY, Moon HJ, Kurata S et al. Purification and cDNA cloning of an antifungal protein from the hemolymph of Holotrichia diomphalia larvae. Biol Pharm Bull 1995; 18:1049–1052.PubMedCrossRefGoogle Scholar
  28. 28.
    Lee YJ, Chung TJ, Park CW et al. Structure and expression of the tenecin 3 gene in Tenebrio molitor. Biochem Biophys Res Commun 1996; 218:6–11.PubMedCrossRefGoogle Scholar
  29. 29.
    Zhang R, Cho HY, Kim HS et al. Characterization and properties of a 1,3-beta-D-glucan pattern recognition protein of Tenebrio molitor larvae that is specifically degraded by serine protease during prophenoloxidase activation. J Biol Chem 2003; 278:42072–42079.PubMedCrossRefGoogle Scholar
  30. 30.
    Lee MH, Osaki T, Lee JY et al. Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect. J Biol Chem 2004; 279:3218–3227.PubMedCrossRefGoogle Scholar
  31. 31.
    Park JW, Je BR, Piao S et al. A synthetic peptidoglycan fragment as a competitive inhibitor of the melanization cascade. J Biol Chem 2006; 281:7747–7755.PubMedCrossRefGoogle Scholar
  32. 32.
    Ju JS, Cho MH, Brade L et al. A novel 40-kDa protein containing six repeats of an epidermal growth factor-like domain functions as a pattern recognition protein for lipopolysaccharide. J Immunol 2006; 177:1838–1845.PubMedGoogle Scholar
  33. 33.
    Lee SY, Kwon TH, Hyun JH et al. In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae. Eur J Biochem 1998; 254:50–57.PubMedCrossRefGoogle Scholar
  34. 34.
    Kwon TH, Kim MS, Choi HW et al. A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae. Eur J Biochem 2000; 267:6188–6196.PubMedCrossRefGoogle Scholar
  35. 35.
    Chasan R, Anderson KV. The role of easter, an apparent serine protease, in organizing the dorsal-ventral pattern of the Drosophila embryo. Cell 1989; 56:391–400.PubMedCrossRefGoogle Scholar
  36. 36.
    Iwanaga S, Lee BL. Recent advances in the innate immunity of invertebrate animals. J Biochem Mol Biol 2005; 38:128–150.PubMedCrossRefGoogle Scholar
  37. 37.
    Murugasu-Oei B, Rodrigues V, Yang X et al. Masquerade: a novel secreted serine protease-like molecule is required for somatic muscle attachment in the Drosophila embryo. Genes Dev 1995; 9:139–154.PubMedCrossRefGoogle Scholar
  38. 38.
    Piao S, Song YL, Kim JH et al. Crystal structure of a clip-domain serine protease and functional roles of the clip domains. EMBO J 2005; 24:4404–4414.PubMedCrossRefGoogle Scholar
  39. 39.
    Piao S, Kim S, Kim JH et al. Crystal structure of the serine protease domain of prophenoloxidase activating factor-I. J Biol Chem 2007; 282:10783–10791.PubMedCrossRefGoogle Scholar
  40. 40.
    Kim MS, Baek MJ, Lee MH et al. A new easter-type serine protease cleaves amasquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae. J Biol Chem 2002; 277:39999–40004.PubMedCrossRefGoogle Scholar
  41. 41.
    Lee HS, Cho MY, Lee KM et al. The pro-phenoloxidase of coleopteran insect, Tenebrio molitor, larvae was activated during cell clump/cell adhesion of insect cellular defense reactions. FEBS Lett 1999; 444:255–259.PubMedCrossRefGoogle Scholar
  42. 42.
    Lee KY, Zhang R, Kim MS et al. A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae. Eur J Biochem 2002; 269:4375–4383.PubMedCrossRefGoogle Scholar
  43. 43.
    Gobert V, Gottar M, Matskevich AA et al. Dual activation of the Drosophila toll pathway by two pattern recognition receptors. Science 2003; 302:2126–2130.PubMedCrossRefGoogle Scholar
  44. 44.
    Kim CH, Kim SJ, Kan H et al. A Three-step Proteolytic Cascade Mediates the Activation of the Peptidoglycan-induced Toll Pathway in an Insect. J Biol Chem 2008; 283:7599–7607.PubMedCrossRefGoogle Scholar
  45. 45.
    Ji C, Wang Y, Guo X et al. A pattern recognition serine proteinase triggers the prophenoloxidase activation cascade in the tobacco hornworm, Manduca sexta. J Biol Chem 2004; 279:34101–34106.PubMedCrossRefGoogle Scholar
  46. 46.
    Wang Y, Jiang H. Interaction of beta-1,3-glucan with its recognition protein activates hemolymph proteinase 14, an initiation enzyme of the prophenoloxidase activation system in Manduca sexta. J Biol Chem 2006; 281:9271–9278.PubMedCrossRefGoogle Scholar
  47. 47.
    Buchon N, Poidevin M, Kwon HM et al. A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway. Proc Natl Acad Sci USA 2009; 106:12442–12447.PubMedCrossRefGoogle Scholar
  48. 48.
    Jang IH, Chosa N, Kim SH et al. A Spätzle-processing enzyme required for toll signaling activation in Drosophila innate immunity. Dev Cell 2006; 10:45–55.PubMedCrossRefGoogle Scholar
  49. 49.
    Roh KB, Kim CH, Lee H et al. Proteolytic cascade for the activation of the insect toll pathway induced by the fungal cell wall component. J Biol Chem 2009; 284:19474–19481.PubMedCrossRefGoogle Scholar
  50. 50.
    Bulet P, Hetru C, Dimarcq JL et al. Antimicrobial peptides in insects; structure and function. Dev Comp Immunol 1999; 23:329–344.PubMedCrossRefGoogle Scholar
  51. 51.
    Bulet P, Cociancich S, Dimarcq JL et al. Insect immunity. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family. J Biol Chem 1991; 266:24520–24525.PubMedGoogle Scholar
  52. 52.
    Gettins PG. Serpin structure, mechanism and function. Chem Rev 2002; 102:4751–4804.PubMedCrossRefGoogle Scholar
  53. 53.
    Gooptu B, Lomas DA. Conformational pathology of the serpins: themes, variations and therapeutic strategies. Annu Rev Biochem 2009; 78:147–176.PubMedCrossRefGoogle Scholar
  54. 54.
    Ligoxygakis P, Roth S, Reichhart JM. A serpin regulates dorsal-ventral axis formation in the Drosophila embryo. Curr Biol 2003; 13:2097–2102.PubMedCrossRefGoogle Scholar
  55. 55.
    Hashimoto C, Kim DR, Weiss LA et al. Spatial regulation of developmental signaling by a serpin. Dev Cell 2003; 5:945–950.PubMedCrossRefGoogle Scholar
  56. 56.
    Scherfer C, Tang H, Kambris Z et al. Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation. Dev Biol 2008; 323:189–196.PubMedCrossRefGoogle Scholar
  57. 57.
    De Gregorio E, Han SJ, Lee WJ et al. An immune-responsive Serpin regulates the melanization cascade in Drosophila. Dev Cell 2002; 3:581–592.PubMedCrossRefGoogle Scholar
  58. 58.
    Ligoxygakis P, Pelte N, Ji C et al. A serpin mutant links Toll activation to melanization in the host defence of Drosophila. EMBO J 2002; 21:6330–6337.PubMedCrossRefGoogle Scholar
  59. 59.
    Tang H, Kambris Z, Lemaitre B et al. A serpin that regulates immune melanization in the respiratory system of Drosophila. Dev Cell 2008; 15:617–626.PubMedCrossRefGoogle Scholar
  60. 60.
    Kanost MR, Jiang H, Yu XQ. Innate immune responses of a lepidopteran insect, Manduca sexta. Immunol Rev 2004; 198:97–105.PubMedCrossRefGoogle Scholar
  61. 61.
    Jiang R, Kim EH, Gong JH et al. Three pairs of protease-serpin complexes cooperatively regulate the insect innate immune responses. J Biol Chem 2009; 284:35652–35658.PubMedCrossRefGoogle Scholar
  62. 62.
    Nash P, Whitty A, Handwerker J et al. Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1. J Biol Chem 1998; 273:20982–20991.PubMedCrossRefGoogle Scholar
  63. 63.
    Jesty J. The kinetics of formation and dissociation of the bovine thrombin antithrombin III complex. J Biol Chem 1979; 254:10044–10050.PubMedGoogle Scholar
  64. 64.
    Han J, Zhang H, Min G et al. A novel Drosophila serpin that inhibits serine proteases. FEBS Lett 2000; 468:194–198.PubMedCrossRefGoogle Scholar
  65. 65.
    Gettins PG, Olson ST. Exosite determinants of serpin specificity. J Biol Chem 2009; 284:20441–20445.PubMedCrossRefGoogle Scholar
  66. 66.
    Chen VC, Chao L, Chao J. Roles of the P1, P2 and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein. J Biol Chem 2000; 275:38457–38466.PubMedCrossRefGoogle Scholar
  67. 67.
    Ferrandon D, Imler JL, Hetru C et al. The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat Rev Immunol 2007; 7:862–874.PubMedCrossRefGoogle Scholar
  68. 68.
    Kan H, Kim CH, Kwon HM et al. Molecular control of phenoloxidase-induced melanin synthesis in an insect. J Biol Chem 2008; 283:25316–25323.PubMedCrossRefGoogle Scholar
  69. 69.
    Wang Y, Jiang H. Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor. Insect Biochem Mol Biol 2007; 37:1015–1025.PubMedCrossRefGoogle Scholar
  70. 70.
    Richer MJ, Keays CA, Waterhouse J et al. The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin. Proc Natl Acad Sci USA 2004; 101:10560–10565.PubMedCrossRefGoogle Scholar
  71. 71.
    Zou Z, Jiang H. Manduca sexta serpin-6 regulates immune serine proteinases PAP-3 and HP8. cDNA cloning, protein expression, inhibition kinetics and function elucidation. J Biol Chem 2005; 280:14341–14348.PubMedCrossRefGoogle Scholar
  72. 72.
    Bjork I, Jackson CM, Jornvall H et al. The active site of antithrombin. Release of the same proteolytically cleaved form of the inhibitor from complexes with factor IXa, factor Xa and thrombin. J Biol Chem 1982; 257:2406–2411.PubMedGoogle Scholar
  73. 73.
    Salvesen GS, Catanese JJ, Kress LF et al. Primary structure of the reactive site of human C1-inhibitor. J Biol Chem 1985; 260:2432–2436.PubMedGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Ji-Won Park
    • 1
  • Chan-Hee Kim
    • 1
  • Jiang Rui
    • 1
  • Keun-HwaPark
    • 1
  • Kyung-Hwa Ryu
    • 1
  • Jun-Ho Chai
    • 1
  • Hyun-Ok Hwang
    • 1
  • Kenji Kurokawa
    • 1
  • Nam-Chul Ha
    • 1
  • Irene Söderhäll
    • 2
  • Kenneth Söderhäll
    • 2
  • Bok Luel Lee
    • 1
  1. 1.National Research Laboratory of Defense Proteins, College of PharmacyPusan National UniversityBusanKorea
  2. 2.Department of Comparative PhysiologyUppsala UniversityUppsalaSweden

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