Inhibition of Diabrotica larval growth by a multicystatin from potato tubers

Abstract

Second instar western corn rootworm larvae have a higher proportion of total proteinase activity which is attributable to cysteine proteinase (92%) than southern corn rootworm larvae (75%). E-64, potato multicystatin (PMC) and tryptic fragments of PMC (T-PMC) are effective inhibitors of gut cysteine proteinase activity, in vitro. The presence of PMC in the diet causes a dose-dependent inhibition of growth in neonate southern corn rootworm and second instar southern corn rootworm and western corn rootworm. Neonate southern corn rootworm and second instar western rootworm have similar sensitivity to the inhibitor (50% inhibition at 25–43.8 μg/cm 2), whereas second instar southern corn rootworm are about 5-fold less sensitive. In contrast to southern corn rootworm larvae, western corn rootworm growth is completely halted by PMC. Long-term exposure of southern corn rootworm larvae to PMC suggests that the larvae become less sensitive to the inhibitor during development. Hen egg cystatin (HEC) and T-PMC are unable to inhibit growth of either species but, in southern corn rootworm, co-feeding of potato carboxypeptidase inhibitor (PCI) with T-PMC causes growth inhibition. Direct measurements of gut cysteine proteinase activity after feeding of the inhibitors indicates that PMC and PCI+T-PMC cause significant inhibition of cysteine proteinase in the gut, whereas HEC, PCI and T-PMC do not. These observations indicate that multicystatins such as PMC may be effective cystatins for use in controlling larvae of Diabrotica species in transgenic plants.