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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 mm 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.

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Proteases in plants carry out an essential role in protein turnover during seed development and germination. Cysteine proteases (EC 3.4.22) are well‐known participants of such processes. Serine proteases (EC 3.4.21), however, are far less reported as protagonists during these events. Here, we investigate this protease class acting in different cowpea seed tissues along their life cycle. In vitro enzymatic assays were performed against N‐CBZ‐Gly‐Gly‐Arg‐MCA, N‐CBZ‐Gly‐Pro‐Arg‐MCA, Z‐Arg‐Arg‐MCA, and Ile‐His‐Pro‐Phe‐MCA fluorimetric substrates, under specific assay conditions. Proteins and proteases were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and SDS‐PAGE‐gelatin, respectively. The proteolytic apparatus operating in cowpea [Vigna unguiculata (L.) Walp.] seeds includes serine endopeptidases in both cotyledons and embryo axes during the whole seed life cycle, mainly from the 12th to the 16th day after pollination and after 12 h of imbibition. The proteolytic activities appeared as bands of molecular masses between 68 and 200 kDa. Studies related to substrate specificity and pattern of inhibition showed that some activities are aprotinin‐sensitive serine endopeptidases, with optimum pH ranging from 8.0 to 9.0 towards N‐CBZ‐Gly‐Gly‐Arg‐MCA and Z‐Arg‐Arg‐MCA, while others are phenylmethanesulphonyl fluoride (PMSF) sensitive, with an optimum pH 7.0, towards Ile‐His‐Pro‐Phe‐MCA. Serine proteases acting in developing and germinating cowpea seeds are both trypsin‐like (EC 3.4.21.4) and chymotrypsin‐like (EC 3.4.21.1) activities.