Characterisation of the mid-gut digestive proteinase activity of the two-spot ladybird ( Adalia bipunctata L.) and its sensitivity to proteinase inhibitors

Abstract

Proteinase activities in the midguts of adult and larval two-spot ladybirds were investigated. Both extracts showed a slightly acidic pH optimum for proteolysis against azocasein (pH 6.0 and pH 5.5 for larvae and adults respectively), which correlated well with the physiological pH of the midgut. The proteolytic activity of the larval midguts, detected by hydrolysis of Z-phe-arg-pNA, was almost totally inhibited by the cysteine proteinase inhibitor E-64. Inhibitors diagnostic for the other mechanistic classes of proteinase had little or no effect on proteolysis. In-gel assays showed that the proteolytic activity was due to three major cysteine proteinases with indicated mol. wts. 23, 30 and 55 kDa. In addition to cysteine proteinases, adult ladybirds also possessed a metallo-proteinase activity. Plant protein inhibitors of serine proteases had little effect on ladybird proteolytic activity, but cystatins (cysteine protease inhibitors) from plant and animal sources showed significant inhibition (up to 90% at 2 μM). The results suggest that ladybird digestion may be affected by cysteine proteinase inhibitors expressed in transgenic plants for pest control, via the tritrophic interaction that occurs between ladybirds, aphids and crop plants.