Midgut proteinase activities in larvae of Anoplophora glabripennis (Coleoptera: Cerambycidae) and their interaction with proteinase inhibitors

Abstract

The major proteinase activities in the larval midgut of a common poplar tree borer, Anoplophora glabripennis, were characterised. Overall digestive capacity, as measured by casein hydrolysis, showed a p H optimum between 10 and 11.5, suggestive of serine endopeptidase activity. Trypsin, chymotrypsin, and chymotrypsin-like activities were detected using specific p-nitroaniiide synthetic substrates and by use of specific serine endopeptidase inhibitors. These activities also showed pH optima in the extreme alkaline range. The absence of cysteine, aspartic, and metallo-endopeptidases were confirmed using class specific proteinase inhibitors. The dominant exopeptidase in the midgut is leucine aminopeptidase with a p H optimum of 7-9. Carboxypeptidase a and b activity were barely detectable. A large range of serine endopeptidase inhibitors were screened and were found to vary widely in their ability to inhibit casein hydrolysis. Potato proteinase inhibitor 1 (a chyrnotrypsin inhibitor) and wheatgerm trypsin inhibitor 1 inhibited particularly effectively in tandem and represent possible candidates for gene transformation to produce plants tolerant to this pest. 0 1996 Wiley-Liss, Inc.

A Putative Serine Protease from Larval Midgut of Red Palm Weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae): Partial Purification and Biochemical Characterization

“…On the other hand, the TLE shows properties similar to other serine proteolytic enzymes from many insects [13] which is revealed highly proteolytic activity at basic pH in the range of 9.5-11 using radial diffusion analysis. Our data are consistent with those reported by [24] who found that the major proteinase activity in the larval midguts of a common poplar tree borer, Anoplophora glabripennis motsch, were showed a pH optimum between 10 to 11.5, suggesting a digestive system based largely on serine-like proteinases.…”

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

“…In this study we characterized the larvae midgut proteolytic enzymes of a obligate vascular feeding species. The range and composition of proteases is similar to those of two sapwood‐feeding species previously reported (Bian et al , 1996; Bozic et al , 2008); endopeptidases are dominated by trypsin and chymotrypsin‐like activities and exopeptidase by leucine aminopeptidase. This similarity exists, despite the concentration of protein and free amino acids available in vascular tissues, especially the phloem, being much higher than in sapwood (Haack & Slansky, 1987; Prins & Kreulen, 1991).…”

“…Supernatants were subsequently used in the assays. Proteolytic activities were measured as previously described (Christeller et al , 1992; Bian et al , 1996). Acetylated ( 14 C) casein was used as a general protein substrate and activity determined as trichloroacetic acid (TCA)‐soluble peptides by scintillation counting.…”

“…These data were re‐examined by Mishra and Sen‐Sarma (1986), who found pepsin, trypsin and expopeptidase activity in the foregut and midgut of Stromatium barbartum larvae and attributed the endopeptdase activities to micro‐organisms. Most recently, studies using the trunk borers (sapwood‐feeders) Anoplophora glabripennis (Bian et al , 1996) and Morimus funereus (Bozic et al , 2003) larvae confirmed the presence of serine proteinase‐based digestive systems and leucine aminopeptidase as the major exopeptidase activity. In this report we have studied the proteolytic activities of a species that, while partly feeding on sapwood tissues, is restricted to wood where vascular tissues are present.…”

Effects of class‐specific, synthetic, and natural proteinase inhibitors on life‐history traits of the cotton bollworm Helicoverpa armigera

“…Trypsin and chymotrypsin activities were assayed using a modified protocol (Bian, Shaw, Han, & Christeller, ) in a total volume of 100 µl in wells of a microtiter plate. Assays contained 85 µl of 0.1 M Tris‐HCl pH 7.8, 5 µl of lumen sample and 10 µl of N ‐benzoyl‐DL‐arginine‐ p ‐nitroanilide and N ‐succinyl‐alanine‐alanine‐proline‐phenylalanine‐ p ‐nitroanilide as specific substrates for trypsin and chymotrypsin, respectively.…”