Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals

Lan Wu, Purdue University

Abstract

Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The results indicated that the major digestive proteinases in the midgut were trypsin-like and chymotrypsin-like serine proteinases. The partial inhibition of proteolytic activity in crude midgut extract toward ($\sp3$H) -methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. The purification scheme gave approximately 30% recovery of the enzyme activities for both proteinases, and a 168-fold increase in specific activity for the trypsin-like enzyme and 630-fold increase for the chymotrypsin-like enzyme. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. The results further suggested the possible presence of a pepstatin A-sensitive proteinase in the midgut. An artificial seed system was developed for S. cerealella. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics. The bioassay results and the model suggest that some proteinaceous proteinase inhibitors could be used as resistance factors to control S. cerealella.

Degree

Ph.D.

Advisors

Shukle, Purdue University.

Subject Area

Entomology

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