A Biochemical study on the types of digestive specific proteases in the ladybird, Cryptolaemus montrouzieri Mulsant (Col.: Coccinellidae) and the effects of different preys on their activity

Document Type : Research Paper

Author

Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran

Abstract

Type and biochemical properties of the digestive proteases were determined using specific substrates and inhibitors in the larvae and the adults of Cryptolaemus montrouzieri and their activity changes were evaluated when fed on the different preys. Rearing of beetles was done in laboratory conditions and they were fed on citrus mealybug. Dissection was done in NaCl medium and the obtained midgut was homogenized and centrifuged. Then, the activity of digestive proteases determined by using the specific substrates and inhibitors by feeding on three hosts. The results showed that just cysteine proteases and exopeptidases were active in the midgut of adults while both serine and cysteine proteases were engaged in the digestion by the larvae. The highest activity of the serine proteases; trypsin, chymotrypsin and elastase, were found in the pH sets of 8, 8-9 and 9, respectively. The optimal pH of cathepsins B and L were determined in 6 and 5 pH values of the larvae and the adults of C. montrouzieri, respectively. The highest activities of digestive larval amino- and carboxypeptidases were measured at pH 7 while these two enzymes showed the highest activity at pH sets of 7 and 6 in the adults, respectively. The specific inhibitors including cystatin, E-64, TLCK, SBTI, TPCK and AEBSF.HCL significantly decreased the activities of specific proteases in the larvae and the adults of C. montrouzieri. No significant differences were observed in the activities of serine proteases, exopeptidases and cathepsin L in the larvae fed on Planococcus citri, Pseudococcus viburni and Toxoptera aurantii but cysteine proteases and aminopeptidases in the adults fed on tea aphid showed the least activity. The difference in proteolytic profile of the larvae and the adults of C. montrouzieri concur the physiological differences in these two developmental stages with the emphasize on serine proteases in the preservation and control of developmental status of digestive tract. In addition, the higher activity of cysteine proteases in the adults may be related to the nature of utilized prey and presence of different proteinaceous components or even glycoperoteins in the preys.

Keywords


Fabrick, J., Behnke, C., Czapla, T., Bala, K., Rao, A.G., Kramer, K.J. and Reeck, G.R. 2002. Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern cornrootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae). Insect Biochemistry and Molecular Biology32: 405-415.
Ferreira, C. and Terra, W.R. 1983. Physical and kinetic properties of a plasma-membranebound P-D-glucosidase (cellobiase) from midgut cells of an insect (Rhynchosciara americana larva). Biochemistry Journal 213: 43–51.
Gholamzadeh-Chitgar, M., Ghadamyari, M. and Ghanbarinejad, R. 2017. Biochemical properties of digestive proteases of melon ladybird, Epilachna chrysomelina (Fabricius) (Col.: Coccinellidae). Journal of Entomological Society of Iran 37: 293-304 [In Persian].
Kanost, M.R. and Clem, R.J. 2011. Insect proteases. In: Gilbert, L.I. (Ed.), Insect Molecular Biology and Biochemistry. Elsevier, Amsterdam, pp. 346–364.
Koo, M. S. and Park, Y. C. 2002. Gut luminal digestive proteinases of adult Lady Beetle, Harmonia axyridis (Coccinellidae: Coleoptera), fed an artificial diet. Journal of Asia-Pacific Entomology 5: 167-173.
Lemos, F. J. A., Campos, F. A. P., Silva, C. P. and Xavier-Filho, J. 1990. Proteinases and amylases of larval midgut of Zabrotes subfasciatus reared on cowpea (Vigna unguiculata) seeds. Entomologia Experimentalis et Applicata56: 219-227.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265–275.
Mehrabadi, M. and Bandani, A. R. 2011. Secretion and formation of perimicrovillar membrane in the digestive system of the Sunn pest, Eurygaster integriceps (Hemiptera: Scutelleridae) in the response of feeding. Archives of Insect Biochemistry and Physiology 78: 190-200.
Nation, J.L. 2008. Insect Physiology and Biochemistry, 2nd edition. CRC Press, London.
Norén, O., Sjostrom, H., Danielsen, E. M., Cowell, G. M. and Skovbjerg, H. 1986. The enzymes of the enterocyte plasma membrane. In P. Desnuelle, H. Sjostrom, and O. Norén (Eds.), Molecular and Cellular Basis of Digestion (pp. 355–365). Amsterdam: Elsevier.
Oppert, B., Morgan, T. D., Hartzer, K., Lenarcic, B., Galesa, K., Brzin, J., Turk, V., Yoza, K., Ohtsubo, K. and Kramer, K. J. 2003. Effects of proteinase inhibitors on digestive proteinases and growth of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology134: 481-490.
Pascual-Ruiz, S., Carrilo, L., Alvarez-Alfageme, F., Ruiz, M., Castanera, P. and Ortego, F. 2009. The effects of different prey regimes on the proteolytic digestion of nymphs of the spined soldier bug, Podisus maculiventris (Hemiptera: Pentatomidae). Bulletin of Entomological Research 99: 487-491.
Rawlings, N. D., Barrett, A. J. and Bateman, A. 2010. MEROPS: the peptidase database. Nucleic Acids Research 38: D227–D233.
Sakurai, H. 1968. Physiological studies on the digestion of coccinellid beetle (Coleoptera: Coccinellidae), with special reference to their food habits. Applied Entomology and Zoology 3: 130-138.
Sorkhabi-Abdolmaleki, S., Zibaee, A., Hosseini, R. and Hoda, H. 2013. Effects of different prey regimes on activities of digestive enzymes in Andrallus spinidens (Hem.: Pentatomidae). Journal of Entomological Society of Iran 33: 57-68 [In Persian].
Tatli, A., Bandani, A. and Naghdi, M. 2010. Study of the digestive protease in the elm leaf beetle Xanthogaleruca luteola (Col., Chrysomylidae). Proceedings of the 19th Iranian Plant Protection Congress, 9-12 Aug., Tehran, Iran, p. 300 [In Persian].
Terra, W.R. and Ferriera, C. 2012. Biochemistry of digestion. In: Gilbert, L.I. (Ed.), Insect Molecular Biology and Biochemistry. Elsevier, pp. 365–418.
Walker, A. J., Ford, L., Majerus, M. E. N., Geoghegan, I. E., Birch, N., Gatehouse, J. A. and Gatehouse, A. M. R. 1998. Characterisation of the mid-gut digestive proteinase activity of the two-spot ladybird (Adalia bipunctata L.) and its sensitivity to proteinase inhibitors. Insect Biochemistry and Molecular Biology 28: 173–180.
Zibaee, A. 2011. Digestive enzymes of large cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae) from developmental and site of activity perspectives. Italian Journal of Zoology 79: 13-26.