© 2008 The Authors Entomologia Experimentalis et Applicata 129: 11–17, 2008 Journal compilation © 2008 The Netherlands Entomological Society 11 DOI: 10.1111/j.1570-7458.2008.00746.x Blackwell Publishing Ltd Binding of Vigna unguiculata vicilins to the peritrophic membrane of Tenebrio molitor affects larval development E. V. Paes 1 , A. F. Uchôa 2 , M. S. T. Pinto, C. P. Silva 3 , K. V. S. Fernandes 1 , A. E. A. Oliveira 1 * & J. Xavier-Filho 1 1 Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-602, Campos dos Goytacazes-RJ, Brazil, 2 Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal-RN, Brazil, and 3 Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, 88040-900, Florianópolis-SC, Brazil Accepted: 17 April 2008 Key words: storage proteins, 7S globulin, chitin, insect toxic proteins, Tenebrionidae, Coleoptera Abstract We investigated the effects of vicilins (7S storage proteins) from Vigna unguiculata (L.) Walp. (Fabaceae), cultivars EPACE-10 [genotype susceptible to the cowpea weevil, Callosobruchus maculatus (Fabricius)] and IT81D-1045 [cowpea weevil-resistant genotype], seeds on Tenebrio molitor L. (Coleoptera: Tenebrionidae) larval development. Toxicity of vicilins was investigated through the incorporation of these proteins in artificial diet offered to the larvae. Binding tests of vicilins to the peritrophic membranes (PM) were carried out by in vitro incubation of PM with solutions of vicilins. Bound proteins were desorbed from PM with 100 m HCl. Desorbed vicilins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoprobing on Western blotting using an anti-vicilin cv. EPACE-10 antibody. The chitin content of the T. molitor PM was evaluated by the Von Wisselingh color test and presence of chitin in the larval PM was confirmed. Bioassays showed that both vicilins from EPACE-10 and IT81D-1045 genotypes were toxic to T. molitor larvae, and in vitro binding assays showed that these seed-storage proteins were capable of binding to the larval PM. Introduction Plants have developed a variety of chemical and physical defense strategies to protect themselves against pests and pathogens. The chemical defense arsenal is composed of secondary metabolic compounds, such as alkaloids, terpenes, phenolic compounds, cyanogenic glycosides, and proteins (Shewry & Lucas, 1997). Examples of defensive proteins include, among others, lectins (Raikhel et al., 1993; Peumans & Van Damme, 1995), chitinases (Chang et al., 1995), digestive enzyme inhibitors (Ryan, 1990; Lu et al., 1998; Pernas et al., 1999), ribosome- inactivating proteins (Stirpe, 2004), and pathogenesis- related proteins (Muthukrishnan et al., 2001). Biochemical studies suggest that variant vicilins (7S storage proteins) from cowpea, (Vigna unguiculata L. (Fabaceae), seeds are also defensive proteins (Sales et al., 1992, 2000; Macedo et al., 1993). It was shown that ingestion of vicilins from an African cowpea weevil-resistant genotype (IT81D-1045) at a level of 2% killed 50% of cowpea weevil, Callosobruchus maculatus (Fabricius) larvae (Macedo et al., 1993). It was also reported that vicilins from non- host seeds of C. maculatus were toxic to the larvae of this bruchid (Yunes et al., 1998). Immunolocalization studies have shown that V. unguiculata seed vicilins from EPACE- 10 and IT81D-1045 bind to the larval C. maculatus midgut epithelium. This ability to bind, together with the low digestibility of these vicilins and their absorption through the midgut, were associated with the deleterious effects of these proteins to this insect (Sales et al., 1992, 2000; Firmino et al., 1996; Fernandes & Xavier-Filho, 1998; Uchoa et al., 2006). Vicilins also bind to the peritrophic membrane (PM) from larvae of Diatraea saccharalis (Fabricius), affecting larval development (Mota et al., 2003). Peritrophic membranes are non-cellular semi-permeable membranes, comprised of a complex mixture of chitin, proteins, glycoproteins, and proteoglycan conjugates (Merzendorfer & Zimoch, 2003). Peritrophic membranes usually have a chitin content of 3–13% (Peters, 1992; Merzendorfer & Zimoch, 2003; Merzendorfer, 2006). *Correspondence: A. E. A Oliveira, Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013- 602, Campos dos Goytacazes-RJ, Brazil. E-mail: elenir@uenf.br