Journal of Comparative Physiology B

, Volume 183, Issue 6, pp 735–747 | Cite as

Proteolytic activity of gut bacteria isolated from the velvet bean caterpillar Anticarsia gemmatalis

  • F. M. Pilon
  • L. E. Visôtto
  • R. N. C. Guedes
  • M. G. A. Oliveira
Original Paper

Abstract

The development of proteinase inhibitors as potential insect control agents has been constrained by insect adaptation to these compounds. The velvet bean caterpillar (Anticarsia gemmatalis) is a key soybean pest species that is well-adapted to proteinase inhibitors, particularly serine-proteinase inhibitors, which are abundant in the caterpillar host. The expression of diverse proteolytic enzymes by gut symbionts may allow the velvet bean caterpillar to circumvent proteinase inhibitors produced by the host plant. In this study, we characterized the proteolytic activity of the four nonpathogenic species of gut bacteria isolated from the velvet bean caterpillar—Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii and Staphylococcus xylosus. Two proteinase substrates, N-α-benzoyl-l-Arg-p-nitroanilide (l-BApNA) and N-α-p-tosyl-l-Arg methyl ester (l-TAME) and five proteinase inhibitors [aprotinin, E-64, ethylenediamine tetraacetic acid (EDTA), pepstatin and N-α-tosyl-l-lysine chloromethyl ketone (TLCK)] as well as CaCl2, pH and temperature profiles were used to characterize the expressed proteolytic activity of these bacterial strains in vitro. Kinetic parameters for proteolytic activity were also estimated. The results of these experiments indicated that serine- and cysteine-proteinase activities were expressed by all four gut bacteria symbionts of the velvet bean caterpillar. The cysteine- and serine-proteinase activities of these gut symbionts were distinct and different from that of gut proteinases of the caterpillar itself. This finding provides support for the potential involvement of gut symbionts in the mitigation of the negative effects of serine-proteinase inhibitors in the velvet bean caterpillar.

Keywords

Gut microbiota Bacteria symbionts Bacteria–insect non-pathogenic interaction Proteinase 

References

  1. Appel HM (1994) The chewing herbivore gut lumen: physicochemical conditions and their impact on plant nutrients, allelochemicals and insect pathogens. In: Bernays EA (ed) Insect–plant interactions, vol 5. CRC, Boca Raton, pp 209–221Google Scholar
  2. Beg QK, Gupta R (2003) Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enz Microbl Technol 32:294–304CrossRefGoogle Scholar
  3. Bignell DE, Eggleton P (1995) On the elevated intestinal pH of higher termites (Isoptera, Termitidae). Insects Socieaux 42:57–69CrossRefGoogle Scholar
  4. Boulter D (1993) Insect pest control by copying nature using genetically engineering crops. Phytochemistry 34:1453–1466PubMedCrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  6. Broadway RM (1995) Are insects resistant to plant proteinase inhibitors? J Insect Physiol 41:107–116CrossRefGoogle Scholar
  7. Broadway RM (1996) Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 32:39–53CrossRefGoogle Scholar
  8. Broadway RM, Duffey SS (1986) Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larval Helithis zea and Spodoptera exigua. J Insect Physiol 32:39–53Google Scholar
  9. Carlini CR, Grossi-de-Sá MF (2002) Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides. Toxicon 40:1515–1539PubMedCrossRefGoogle Scholar
  10. Cooper J, Dobson H (2007) The benefits of pesticides to mankind and the environment. Crop Prot 26:1337–1348CrossRefGoogle Scholar
  11. D`Avila-Levy CM, Souza RF, Gomes RC, Vermelho AB, Branquinha MH (2003) A novel extracellular calcium-dependent cysteine proteinase from Crithidia deanei. Arch Biochem Biophys 420:1–8CrossRefGoogle Scholar
  12. Dillon RJ, Dillon VM (2004) The gut bacteria of insects: non-pathogenic interactions. Annu Rev Entomol 49:71–92PubMedCrossRefGoogle Scholar
  13. Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278PubMedCrossRefGoogle Scholar
  14. Gatehouse JA, Gatehouse AMR (1998) Identifying proteins with insecticidal activity: use of encoding genes to produce insect-resistant transgenic crops. Pestic Sci 52:165–175CrossRefGoogle Scholar
  15. Hilder VA, Boulter D (1999) Genetic engineering of crop plants for insect resistance—a critical review. Crop Prot 18:177–191CrossRefGoogle Scholar
  16. Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 330:160–163CrossRefGoogle Scholar
  17. Hummel BCW (1959) A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can J Biochem Physiol 37:1393–1399PubMedCrossRefGoogle Scholar
  18. Indiragandhi P, Anandham R, Madhalyan M, Poonguzhali S, Kim GH, Saravanan VS, Sa T (2007) Cultivable bacteria associated with larval gut of prothiofos-resistant, prothiofos-susceptible and field-caught populations of diamondback moth, Plutella xylostella and their potential for antagonism towards entomopathogenic fungi and host insect nutrition. J Appl Microbiol 103:2664–2675PubMedCrossRefGoogle Scholar
  19. Indiraghandi P, Anandham R, Madhalyan M, Sa TM (2008) Characterization and plant growth-promoting traits of bacteria isolated from larval guts of diamondback moth Plutella xylostella (Lepidoptera: Plutellidae). Curr Microbiol 56:327–333CrossRefGoogle Scholar
  20. Indiraghandi P, Yoon C, Yang JO, Cho S, Sa TM (2010) Microbial communities in the developmental stages of B and Q biotypes of sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). J Korean Soc Appl Biol Chem 53:605–617CrossRefGoogle Scholar
  21. Institute SAS (2008) SAS/STAT user’s guide. SAS Institute, CaryGoogle Scholar
  22. Isman MB (2006) Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu Rev Entomol 51:45–66PubMedCrossRefGoogle Scholar
  23. Jongsma MA, Bolter C (1997) The adaptation of insects to plant protease inhibitors. J Insect Physiol 43:885–895PubMedCrossRefGoogle Scholar
  24. Jongsma MA, Bakker PL, Peters J, Bosch D, Stickema WJ (1995) Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 92:8041–8045PubMedCrossRefGoogle Scholar
  25. Joo HS, Kumar CG, Park GC, Kim KT, Paik SR, Chang CS (2002) Optimization of the production of an extracellular alkaline protease from Bacillus horikoshii. Proc Biochem 38:155–159CrossRefGoogle Scholar
  26. Kawalec M, Potempa JL, Moon JL, Travis J, Murray BE (2005) Molecular diversity of a putative virulence factor: purification and characterization of isoforms of an extracellular serine glutamyl endopeptidase of Enterococcus faecalis with different enzymatic activities. J Bacteriol 187:266–275PubMedCrossRefGoogle Scholar
  27. Laemmli UK (1970) Cleavage os structural protein during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  28. Marinho-Prado JS, Lourenção AL, Oliveira JA, Guedes RNC, Oliveira MGA (2011) Survival and feeding avoidance of the eucalyptus defoliator Thyrinteina arnobia exposed to the proteinase inhibitor berenil. J Appl Entomol 135:763–770CrossRefGoogle Scholar
  29. Matsumura F (2004) Contemporary issues on pesticide safety. J Pestic Sci 29:299–303CrossRefGoogle Scholar
  30. Matthews GA (2008) Attitudes and behaviors regarding use of crop protection products—a survey of more than 8500 smallholders in 26 countries. Crop Prot 27:834–846CrossRefGoogle Scholar
  31. Mendonça EG, Oliveira MGA, Visôtto LE, Guedes RNC (2012) Midgut cysteine-proteinase activity in the velvet bean caterpillar [Anticarsia gemmatalis (Hübner)]. J Pest Sci 85:117–123CrossRefGoogle Scholar
  32. Metcalf RL (1980) Changing role of insecticides in crop protection. Annu Rev Entomol 25:219–256CrossRefGoogle Scholar
  33. Mohamed SA, Fahmy AS, Mohamed TM, Hamdy SM (2005) Proteases in egg, miracidium and adult of Fasciola gigantic. Characterization of serine and cysteine proteases from adult. Comp Biochem Physiol B 142:192–200PubMedCrossRefGoogle Scholar
  34. Moreira LF, Campos WG, Ribeiro FR, Guedes RNC, Oliveira MGA (2011) Survival and developmental impairment induced by the trypsin inhibitor bis-benzamidine in the velvet bean caterpillar (Anticarsia gemmatalis). Crop Prot 30:1285–1290CrossRefGoogle Scholar
  35. Nicholson GM (2007) Fighting the global pest problem. Toxicon 49:413–422PubMedCrossRefGoogle Scholar
  36. Oliveira MGA, De Simone SG, Xavier LP, Guedes RNC (2005) Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol B 140:369–380PubMedCrossRefGoogle Scholar
  37. Pilon AM, Oliveira MGA, Guedes RNC (2006) Protein digestibility, protease activity, and post-embryonic development of the velvet bean caterpillar (Anticarsia gemmatalis) exposed to the trypsin-inhibitor benzamidine. Pestici Biochem Physiol 86:23–29CrossRefGoogle Scholar
  38. Pinto-Tomás AA, Sittenfeld A, Uribe-Lorío L, Chavarría F, Mora M, Janzen DH, Goodman RM, Simon HM (2011) Comparison of midgut bacterial diversity in tropical caterpillars (Lepidoptera: Saturniidae) fed o different diets. Environ Entomol 40:1111–1122PubMedCrossRefGoogle Scholar
  39. Pompermayer P, Lopes AR, Parra JRP, Terra WR, Silva-Filho M (2001) Effects of soybean proteinase inhibitor on development, survival and reproductive potential of the sugarcane borer, Diatraea saccharalis. Entomol Exp Appl 99:79–85CrossRefGoogle Scholar
  40. Rao MB, Aparna MT, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635PubMedGoogle Scholar
  41. Reeck GR, Oppert B, Denton M, Kanost M, Baker JE, Kramer KJ (1999) Insect proteinases. In: Truk V (ed) Proteases: new perspectives. Birkhausser, Boston, pp 125–148CrossRefGoogle Scholar
  42. Rosell G, Quero C, Coll J, Guerrero A (2008) Biorational insecticides in pest management. J Pestic Sci 33:103–121CrossRefGoogle Scholar
  43. Shaw E, Mares-Guia M, Cohen W (1965) Evidence for na active center histidine in trypsin with the use of a specific reagent, 1-chloro-3-tosylamido-7-amino-2-heptanone, the chloromethyl ketone derived from N-alpha-tosyl-l-lysine. Biochemistry 4:2219–2224CrossRefGoogle Scholar
  44. Silva LB, Reis AP, Pereira EJG, Oliveira MGA, Guedes RNC (2010a) Altered cysteine-proteinase activity in insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais. Comp Biochem Physiol B 157:80–87PubMedCrossRefGoogle Scholar
  45. Silva LB, Reis AP, Pereira EJG, Oliveira MGA, Guedes RNC (2010b) Partial purification and characterization of trypsin-like proteinases from insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais. Comp Biochem Physiol B 155:12–19PubMedCrossRefGoogle Scholar
  46. Silva-Lopez RE, De Simone SG (2004) Leishmania (Leishmania) amazonensisi: purification and characterization of a promastigote serine protease. Exp Parasitol 107:173–182PubMedCrossRefGoogle Scholar
  47. Sipos T, Merkel JR (1970) Na effect of calcium ions on the activity, heat stability, and structure of trypsin. Biochemistry 9:2766–2775PubMedCrossRefGoogle Scholar
  48. Stotz HU, Kroymann J, Mitchell-Olds T (1999) Plant–insect interactions. Cur Opin Plant Biol 2:268–272CrossRefGoogle Scholar
  49. Systat 2008. SigmaPlot v.10 user’s guide. Systat Software, ChicagoGoogle Scholar
  50. Terra WR, Ferreira C (2005) Biochemistry and digestion. In: Gilbert LI, Iatrou K, Gill SS (eds) Comprehensive molecular insect science, vol 4. Pergamon, New York, pp 171–224CrossRefGoogle Scholar
  51. Vajda T, Garai A (1981) Comparison of the effect of calcium (II) and manganese (II) ions on trypsin autolysis. J Inorg Biochem 15:307–315PubMedCrossRefGoogle Scholar
  52. Valasaki K, Staikou A, Theodorou LG, Charamopoulou V, Zacharaki P, Papamichael EM (2008) Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest. Biores Technol 99:5804–5813CrossRefGoogle Scholar
  53. Visôtto LE, Oliveira MGA, Guedes RNC, Ribon AOB, Good-God PIV (2009a) Contribution of gut bacteria to digestion and development of the velvet bean caterpillar, Anticarsia gemmatalis. J Insect Physiol 55:185–191PubMedCrossRefGoogle Scholar
  54. Visôtto LE, Oliveira MGA, Ribon AOB, Mares-Guia TR, Guedes RNC (2009b) Characterization and identification of proteolytic bacteria from the gut of the velvet bean caterpillar (Lepidoptera: Noctuidae). Environ Entomol 38:1078–1085PubMedCrossRefGoogle Scholar
  55. Walenciak O, Zwisler W, Gross EM (2002) Influence of myriophyllum spicatum-derived tannins on gut microbiota of its herbivore Acenria ephemerella. J Chem Ecol 28:2045–2056PubMedCrossRefGoogle Scholar
  56. Walker AJ, Glen DM, Shewry PR (1999) Bacteria associated with the digestive system of the slug Deroceras reticulatumi are not required for protein digestion. Soil Biol Biochem 31:1387–1394CrossRefGoogle Scholar
  57. Wang SL, Chen YH, Wang CL, Yen YH, Chern MK (2005) Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigates in a shrimp and crab shell powder medium. Enz Microb Technol 36:660–665CrossRefGoogle Scholar
  58. Xavier LP, Oliviera MGA, Guedes RNC, Santos AV, De Simone SG (2005) Trypsin-like activity of membrane-bound midgut proteases from Anticarsia gemmatalis (Lepidoptera: Noctuidae). Eur J Entomol 102:147–153Google Scholar
  59. Zaspel JM, Hoy MA (2008) Microbial diversity associated with the fruit-piercing and blood-feeding moth Calyptra thalictri (Lepidoptera: Noctuidae). An Entomol Soc Am 101:1050–1055CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • F. M. Pilon
    • 1
  • L. E. Visôtto
    • 1
    • 2
  • R. N. C. Guedes
    • 3
    • 4
  • M. G. A. Oliveira
    • 1
    • 4
  1. 1.Departamento de Bioquímica e Biologia MolecularUniversidade Federal de ViçosaViçosaBrazil
  2. 2.Instituto de Ciências AgráriasUniversidade Federal de Viçosa, Campus Rio ParanaíbaRio ParanaíbaBrazil
  3. 3.Departamento de EntomologiaUniversidade Federal de ViçosaViçosaBrazil
  4. 4.Instituto Nacional de Ciência e Tecnologia em Interações Planta-PragaUniversidade Federal de ViçosaViçosaBrazil

Personalised recommendations