Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera: Bruchidae) and the induction of α–amylases in response to different diets

Abstract

Zabrotes subfasciatus larvae possess three α–amylase isoforms as determined by in gel assays following SDS–PAGE. The two minor isoforms present lower electrophoretic mobility than the major form, and seem to occur as a heterodimer. When developed inside Vigna unguiculata (cowpea) seeds, fourth instar larvae have minor quantities of the slow-migrating forms, but when reared on seeds of Phaseolus vulgaris (common bean) or Phaseolus lunatus, the two slow-migrating forms are expressed in higher amounts, while activity of the major form was independent of the host seed. Larvae developing inside cowpea seeds at the beginning of the fourth instar were fed on flour from cotyledons of cowpea or common bean. Larvae fed on the common bean flour started to express the dimer in higher amounts when compared with the control larvae fed on cowpea flour. In an attempt to correlate differences between starch granules and the induction of α–amylases, a detailed study on the digestive process of the granules was conducted. Incorporation of purified starch granules into artificial diets did not induce the two minor α–amylases. The in vitro hydrolysis rates of purified granules and the pattern of dextrins liberated by the different α–amylases were similar for the two legume species. The starch granules enter the midgut extensively damaged, which may facilitate the access to the more susceptible parts of the granules to enzymatic attack.

Introduction

Bruchid beetles are major pests of stored legume grains throughout the world (Southgate, 1979). Among the most important bruchid pests, the Mexican bean weevil Zabrotes subfasciatus is of particular importance, because it is capable of infesting a relatively broad spectrum of legume species, in particular two of the most important legumes utilized for human consumption, the common bean (Phaseolus vulgaris) and the cowpea (Vigna unguiculata). In a recent screening of the digestive carbohydrases of Z. subfasciatus, we demonstrated that its larvae were well equipped with a suitable arsenal of enzymes for the digestion of the seed starch (Silva et al., 1999). It is particularly important that Z. subfasciatus larvae have the ability to modulate the concentration of α–glucosidase and α–amylase when reared on different legume seeds, secreting a higher amount of two amylase isoforms and one α–glucosidase when developing on P. vulgaris compared with V. unguiculata seeds.

In spite of the importance of starch utilization by granivorous insects, for example, as targets for insect control (Shade et al., 1994, Schroeder et al., 1995), the process of starch granule digestion is still overlooked. Raw starch granules are the natural substrate and so precaution is necessary when extrapolating information from data based on gelatinized starch (Baker and Woo, 1992, Baker et al., 1992). As for most insects, information on the starch digestion process in bruchid species is very scarce, in contrast to the knowledge about the kinetic aspects of the enzymes involved in this process (Podoler and Applebaum, 1971, Lemos et al., 1990, Baker and Woo, 1992, Terra and Ferreira, 1994, Grossi-de-Sá and Chrispeels, 1997).

In order to increase our knowledge of the process by which larvae of Z. subfasciatus adapt to different host seeds, we investigated the possibility that differences in the starch granules found in the common bean and cowpea may be correlated with differences in α–amylase patterns seen when the larvae were reared on these two legume species. Although a direct role of the starch granules in inducing different amylases in Z. subfasciatus was not confirmed, our study provides a detailed account of the digestive process of starch utilization by this bruchid larva.