Larvae removed from seeds of V. unguiculata were transferred to a cavity produced in the compacted mass of flour in one half of the gelatin capsule, at a ratio of three larvae per capsule. Following this, the two halves of the capsules were carefully joined together in order to permit the feeding ABT 199 movements of the larvae and maintained in the dark. Controls were used in which only FITC was mixed with seed flour at the concentration of 2.0% (w/w) in order to assess the level of FITC absorption. Capsules containing only cowpea flour

were used as controls to evaluate auto-fluorescence of the internal organs. Larvae were left to complete their metamorphosis until emergence of adults. In order to visualize and document the presence of labelled vicilins by microscopy from gonads and eggs, fresh portions were mounted on glass slides and visualized using a laser Confocal microscope (Leica DMI6000 B Microscope). Vicilin–FITC fed and mated females 3-days after emergence were transferred to glass vials and maintained during 24 h inside an incubator at 28 °C and 70% RH and without access to males. After this time, the eggs laid on cowpea seeds were removed with a fine needle, placed in a 1.5 mL tube and homogenized (50 eggs/150 μL) in 250 mM NaCl at 4 °C. The homogenate was centrifuged at

15,000 × g for 15 min at 4 °C and the proteins in the supernatant were fractionated by SDS–PAGE as previously described. Virgin vicilin–FITC fed males and control females

check details that copulated with some of those males were dissected and their genitalia and fat bodies were collected. Following collection, some genital tracts were freshly prepared for confocal microscopy and pooled genitalia were homogenized in water using a hand-held Potter–Elvehjem homogenizer immersed in ice. Tissue homogenates were centrifuged at 15,000 × g for 30 min at 4 °C and the supernatants were used for protein determination and fractionation by SDS–PAGE as previously described. Preparative gel electrophoresis (SDS–PAGE) comprising ca 30 μg of proteins from Phosphatidylinositol diacylglycerol-lyase C. maculatus whole egg homogenates and 50 μg of protein from genitalia of both males and females were run as above and stained with Coomassie Blue. Protein bands with Rf similar to peptides recognized by the anti-vicilin antibody (see Souza et al., 2010) were then located on the preparatory gels and excised manually. Gel slices were distained (0.1 M ammonium bicarbonate and 40% acetonitrile), dehydrated (100% acetonitrile) and dried in a speed-vac. Protein digestion was performed as described by Demartini et al. (2011). The tryptic peptides collected after digestion were analyzed by reversed-phase HPLC coupled with tandem mass spectrometry (LC–MS/MS) performed in an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF Micro™, Micromass, Waters, Milford, United States).