International Journal of Tropical Insect Science

, Volume 34, Issue 3, pp 172–178 | Cite as

Gut protease profiles of different instars of Helicoverpa armigera (Lepidoptera: Noctuidae)

  • Lunminlal Kipgen
  • Kamal K. AggarwalEmail author
Research Paper


Pest control strategies against Helicoverpa armigera (Hübner) using protease inhibitors have relied on the gut protease profile of the later larval stages of the insect, with serine proteases being considered predominant. Little is known about the gut protease profile of early larval instars. Therefore, the aim of the present study was to detect the levels of gut protease activities in the third-, fourth-, fifth- and late fifth-instar larvae of H. armigera reared on an artificial diet using specific substrates and inhibitors. The analysis of the gut protease profiles of different instars revealed different levels of protease activities at different instar stages. Significant variations were also observed in the specific activities of trypsin, chymotrypsin, cysteine protease, carboxypeptidase-A and aminopeptidase-N across the instars. In general, the activities of the proteases increased from the third to the fifth instar and then decreased at the onset of pupation in the late fifth instar. Proteolytic activity was optimal at pH 12 for gut extracts from the third-, fourth- and fifth-instar larvae. Bioassays with phenylmethylsulphonyl fluoride (PMSF), a serine-cysteine protease inhibitor, revealed high mortality, and that with sodium ethylenediaminetetraacetic acid (EDTA-Na), a metalloprotease inhibitor, also showed retarded larval growth. The inhibition induced by 0.05% PMSF and 0.05% EDTA-Na in combination was similar to that induced by 0.1% PMSF alone.

Key words

gut protease profile Helicoverpa armigera larval instars pest control protease inhibition 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Angelucci C., Barrett-Wilt G. A., Hunt D. F., Akhurst R. J., East P. D., Gordon K. H. J. and Campbell P. M. (2008) Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis. Insect Biochemistry and Molecular Biology 38, 685–696.CrossRefGoogle Scholar
  2. Arnon R. (1970) Proteolytic enzymes. Methods in Enzymology 19, 226–244.CrossRefGoogle Scholar
  3. Bergmeyer H. U., Gawehn K. and Grassl M. (1974) Enzymes as biochemical reagents, pp. 436–437. In Methods of Enzymatic Analysis, Vol. 1 (edited by H. U. Bergmeyer). Academic Press, New York.Google Scholar
  4. Bown D. P., Wilkinson H. S. and Gatehouse J. A. (1997) Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochemistry and Molecular Biology 27, 625–638.CrossRefGoogle Scholar
  5. Bown D. P., Wilkinson H. S. and Gatehouse J. A. (1998) Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression. Insect Biochemistry and Molecular Biology 28, 739–749.CrossRefGoogle Scholar
  6. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.CrossRefGoogle Scholar
  7. Browne L. B. and Raubenheimer D. (2003) Ontogenetic changes in the rate of ingestion and estimates of food consumption in fourth and fifth instar Helicoverpa armigera caterpillars. Journal of Insect Physiology 49, 63–71.CrossRefGoogle Scholar
  8. Chougule N. P., Giri A. P., Sainani M. N. and Gupta V. S. (2005) Gene expression patterns of Helicoverpa armigera gut proteases. Insect Biochemistry and Molecular Biology 35, 355–367.CrossRefGoogle Scholar
  9. Damle M. S., Giri A. P., Sainani M. N. and Gupta V. S. (2005) Higher accumulation of proteinase inhibitors in flowers than leaves and fruits as a possible basis for differential feeding preference of Helicoverpa armigera on tomato (Lycopersicon esculentum Mill, Cv. Dhanashree). Phytochemistry 66, 2659–2667.CrossRefGoogle Scholar
  10. Filippova I. Y., Lysogorskaya E., Oksenoit E., Rudenskaya G. and Stepanov V. (1984) L-Pyroglutamyl-l-phenyl-alanyl-l-leucine-p-nitroanilide - a chromogenic substrate for thiol proteinase assay. Analytical Biochemistry 143, 293–297.CrossRefGoogle Scholar
  11. McCaffery A. R. (1998) Resistance to insecticides in heliothine Lepidoptera: a global view. Philosophical Transactions of the Royal Society of London Series B: Biological Sciences 353, 1735–1750.CrossRefGoogle Scholar
  12. Nagarkatti S. and Prakash A. (1974) Rearing of Heliothis armigera (Hübner) on an artificial diet. Technical Bulletin of the Commonwealth Institute of Biological Control, Bangalore 17, 169–173.Google Scholar
  13. Oppert B., Morgan T. D. and Kramer K. J. (2004) Inhibitor strategies to control coleopteran pests. International Congress Series 1275, 149–156.CrossRefGoogle Scholar
  14. Özgür E., Yücel M. and Öktem H. A. (2009) Identification and characterization of hydrolytic enzymes from the midgut of the cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae). Turkish Journal of Agriculture and Forestry 33, 285–294.Google Scholar
  15. Patankar A. G., Giri A. P., Harsulkar A. M., Sainani M. N., Deshpande V. V., Ranjekar P. K. and Gupta V. S. (2001) Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest. Insect Biochemistry and Molecular Biology 31, 453–464.CrossRefGoogle Scholar
  16. Pauchet Y., Muck A., Svatos A., Heckel D. G. and Preiss S. (2008) Mapping the larval midgut lumen proteome of Helicoverpa armigera, a generalist herbivorous insect. Journal of Proteome Research 7, 1629–1639.CrossRefGoogle Scholar
  17. Peleiderer G. (1970) Proteolytic enzymes, pp. 514–521. In Methods in Enzymology, Vol. 19 (edited by G. E. Perlmann and L. Lorand). Academic Press, New York.Google Scholar
  18. Secades P. and Guijarro J. (1999) Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production. Applied and Environmental Microbiology 65, 3969–3975.PubMedPubMedCentralGoogle Scholar
  19. Senthilkumar R., Cheng C. P. and Yeh K. W. (2010) Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco. Plant Biotechnology Journal 8, 65–75.CrossRefGoogle Scholar
  20. Tamhane V. A., Chougule N., Giri A., Dixit A., Sainani M. and Gupta V. (2005) In vivo and in vitro effect of Capsicum annuum proteinase inhibitors on Helicoverpa armigera gut proteinases. Biochimica et Biophysica Acta (BBA) - General Subjects 1722, 156–167.CrossRefGoogle Scholar
  21. Tamhane V. A., Giri A. P., Sainani M. N. and Gupta V. S. (2007) Diverse forms of Pin-II family proteinase inhibitors from Capsicum annuum adversely affect the growth and development of Helicoverpa armigera. Gene 403, 29–38.CrossRefGoogle Scholar
  22. Wirnt R. (1974a) Chymotrypsin, measurements with n-benzoyl-l-tyrosine ethyl ester as substrate, pp. 1006–1012. In Methods of Enzymatic Analysis: Samples, Reagents, Assessment of Results, Vol. 2 (edited by H. U. Bergmeyer). Verlag Chemie, Weinheim.Google Scholar
  23. Wirnt R. (1974b) Trypsin, measurements with N-α-p- toluenesulfonyl-l-arginine methyl ester as substrate, pp. 1013–1024. In Methods of Enzymatic Analysis: Samples, Reagents, Assessment of Results, Vol. 2 (edited by H. U. Bergmeyer). Verlag Chemie, Weinheim.Google Scholar
  24. Yang Y., Chen H., Wu Y., Yang Y. and Wu S. (2007) Mutated cadherin alleles from a field population of Helicoverpa armigera confer resistance to Bacillus thuringiensis toxin Cry1Ac. Applied and Environmental Microbiology 73, 6939–6944.CrossRefGoogle Scholar

Copyright information

© ICIPE 2014

Authors and Affiliations

  1. 1.University School of BiotechnologyGuru Gobind Singh Indraprastha UniversityDwarkaIndia

Personalised recommendations