Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

Abstract

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin ( M r 26 000) and four trypsins (T1–T4) with the following properties: T1, K m 0.3 μM, M r 70 000 ; T2, K m 0.4 μM, M r 67 000; T3, K m 2.4 μM, M r 29 000; T4, K m 15 μM, M r 17 000. Diet-fed larvae have a chymotrypsin ( M r 26 000) and a major trypsin ( K m 2.9 μM, M r 29 000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K m values) and a putative decreased affinity for PIs.