RESUMO
Schinus terebinthifolius, Raddi, has been extensively studied due to its anti-inflammatory and antibiotic properties. S. terebinthifolius was also toxic to some insects, however little has been explored about the nature of its insecticide compounds or the toxicity of this plant to insect species. In this work, we investigate the toxicity of S. terebinthifolius seed flour against the insect C. maculatus. S. terebinthifolius seed flour interfered with the post hatch development of the C. maculatus larvae, decreasing larval survival, mass and length. Using DEAE-cellulose chromatography, five protein fractions were isolated, a non-retained fraction (NRF) and four retained fractions, eluted with 0.25, 0.5, 0.7 and 1.0 M NaCl. Proteins with varying molecular masses were observed in all fractions. The majority protein bands were identified by mass spectrometry analysis and among the main identified proteins are 11S globulins (such glycinin), lipoxygenase, chitinases, 7S globulins (vicilins, canavalin and ß conglycinin), annexin, catalase and sucrose binding protein. All DEAE-protein fractions were toxic to the insect, interfering with the post hatch larval development and survival. Decreases greater than 90% were observed in the larval mass and length at 20 days after oviposition (DAO) for larvae raised on diet containing 0.5% of some fractions. Alterations in the level of proteins, glucose and in the activity of the enzymes lipases and cysteine proteases were also detected in these larvae. Our results show that seeds of S. terebinthifolius have an arsenal of toxic proteins with potential for the control of the insect C. maculatus.
Assuntos
Anacardiaceae , Besouros , Vigna , Gorgulhos , Animais , Feminino , Farinha , Larva , Sementes/química , Gorgulhos/metabolismoRESUMO
Vicilins are related to cowpea seed resistance toward Callosobruchus maculatus due to their ability to bind to chitinous structures lining larval midgut. However, this binding mechanism is not fully understood. Here, we identified chitin binding sites and investigated how in vitro and in silico chemical modifications interfere with vicilin chitin binding and insect toxicity. In vitro assays showed that unmodified vicilin strongly binds to chitin matrices, mainly with acetylated chitin. Chemical modifications of specific amino acids (tryptophan, lysine, tyrosine), as well as glutaraldehyde cross-linking, decreased the evaluated parameters. In silico analyses identified at least one chitin binding site in vicilin monomer, the region between Arg208 and Lys216, which bears the sequence REGIRELMK and forms an α helix, exposed in the 3D structure. In silico modifications of Lys223 (acetylated at its terminal nitrogen) and Trp316 (iodinated to 7-iodine-L-tryptophan or oxidized to ß-oxy-indolylalanine) decreased vicilin chitin binding affinity. Glucose, sucrose, and N-acetylglucosamine also interfered with vicilin chitin binding affinity.
Assuntos
Quitina/metabolismo , Besouros/metabolismo , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Quitina/química , Besouros/química , Besouros/efeitos dos fármacos , Simulação por Computador , Larva/química , Larva/efeitos dos fármacos , Larva/metabolismo , Ligação Proteica , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Vigna/química , Vigna/genética , Vigna/metabolismoRESUMO
The seed coat is an external tissue that participates in defense against insects. In some nonhost seeds, including Albizia lebbeck, the insect Callosobruchus maculatus dies during seed coat penetration. We investigated the toxicity of A. lebbeck seed coat proteins to C. maculatus. A chitin-binding protein fraction was isolated from seed coat, and mass spectrometry showed similarity to a C1 cysteine protease. By ELM program an N-glycosylation interaction motif was identified in this protein, and by molecular docking the potential to interact with N-acetylglucosamine (NAG) was shown. The chitin-binding protein fraction was toxic to C. maculatus and was present in larval midgut and feces but not able to hydrolyze larval gut proteins. It did not interfere, though, with the intestinal cell permeability. These results indicate that the toxicity mechanism of this seed coat fraction may be related to its binding to chitin, present in the larvae gut, disturbing nutrient absorption.