Vicilins (7S storage globulins) of cowpea (Vigna unguiculata) seeds bind to chitinous structures of the midgut of Callosobruchus maculatus (Coleoptera: Bruchidae) larvae

Abstract

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insects midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae.

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

“…These Kunitz-type inhibitors also showed an unusual property of interaction with a chitin matrix column. This chitin binding behavior could explain the greater deleterious effect on C. maculatus larvae, since these bruchid larvae possess chitin components of the peritrophic membrane (or equivalent structures) in the midgut [63]. Here, the occurrence of a high inhibition on CpaTI in vitro activity, similar to that of ApTI and DMTI-II, indicates that it may be possible to use CpaTI as an insecticidal agent.…”

Variant vicilins from a resistant Vigna unguiculata lineage (IT81D-1053) accumulate inside Callosobruchus maculatus larval midgut epithelium

“…It has been demonstrated that variant vicilins of cowpea seeds are the main resistance factor present in some African cowpea genotypes, protecting the seeds against attack by the bean beetle C. maculatus (Macedo et al, 1993;Sales et al, 2001). The available evidence suggest that the toxic effects of the variant vicilins, purified from the resistant cowpea variety IT81D-1045, towards larval C. maculatus are linked to resistance of these vicilins to hydrolysis (Sales et al, 1992), chitin-binding activity and binding to the midgut microvilli leading to antibiosis, putatively due to interference in the physiology of the midgut cells (Firmino et al, 1996; Sales et al (2001) demonstrated through immunostaining experiments that variant vicilins from the IT81D-1045 variety bound with higher affinity to midgut cell membrane preparations than susceptible vicilins. Furthermore, immuno-electron localization of vicilins showed that the luminal gut wall surface was strongly labelled.…”

“…However, careful inspections of the published micrographs revealed that vicilins derived from the resistant variety IT81D-1045 can be seen not only on the surface of intestinal epithelial cells, but also within these cells and also in fat body cells (see Fig. 5 in Sales et al, 2001). More recent findings have demonstrated that vicilin molecules are absorbed by the midgut epithelium of C. maculatus larvae (Uchôa et al, 2006;Souza et al, 2010).…”

“…As vicilin molecules are heterogeneous due to the fact that their subunits are encoded by multigene families and post-translational modifications can occur, some forms of vicilins may have higher affinity for their receptors. It is possible that this binding power, associated to a proteolysis resistance (Sales et al, 2001) may interfere with the dissociation of the complexes in endosome vesicles. Further investigations of the presence of vicilin-binding molecule(s) in the midgut tissue and the precise endocytic route involved are already under investigation in our laboratories and this will hopefully increase our comprehension of the mechanism of action of cowpea variant vicilins and other 7S globulins that bean beetles are not adapted.…”

“…The defensive role of variant vicilins against the cowpea beetle was first reported from the genotype IT81D-1045, that is derived from the cultivar TVu 2027 and is also resistant to C. maculatus (Macedo et al, 1993). The mechanism of action of the variant vicilins appears to be correlated to their resistance to proteolysis by digestive peptidases and their binding affinity to C. maculatus midgut epithelial cell surfaces (Firmino et al, 1996;Sales et al, 2001).…”