Proteins involved in microapocrine secretion in Spodoptera frugiperda caterpillar (Lepidoptera)

A região anterior do intestino médio de Lepidoptera apresenta uma secreção microapócrina de enzimas digestivas com vesículas migrando pelo interior das microvilosidades. Essas vesículas brotam das microvilosidades intestinais como vesículas de membrana dupla e são descarregadas dentro do lúmen. O objetivo desse trabalho foi identificar as proteínas secretadas e aquelas envolvidas na maquinaria secretória microapócrina em Spodoptera frugiperda. Para isso, vesículas microapócrinas foram preparadas e usadas para a produção de anticorpo policlonal. Esse anticorpo foi utilizado para varrer uma biblioteca de expressão de cDNA do intestino médio de S. frugiperda. Também obtivemos um transcriptoma por pirosequenciamento de uma biblioteca de cDNA proveniente dos transcritos do intestino médio do mesmo inseto. Os clones positivos da varredura foram sequenciados, montados e submetidos a um BLASTN contra as sequências obtidas pelo pirosequenciamento, o que resultou na extensão dessas sequências. Usamos ainda as sequências geradas pelo pirosequenciamento para reanalisar sequências de proteínas presentes nas membranas microvilares, que tinham sido obtidas anteriormente em nosso laboratório (Ferreira et al., 2007). A reanálise das sequências de proteínas microvilares gerou 66 proteínas preditas. Dessas, 18 foram consideradas contaminantes de outros compartimentos celulares e 48 associadas às membranas microvilares. A análise das sequências obtidas das vesículas microapócrinas gerou 50 proteínas preditas que podem ser secretadas por essa rota. As sequências encontradas tanto em membrana microvilar quanto em vesículas microapócrinas podem ser classificadas em 8 grupos, de acordo com sua função: (1) enzimas digestivas; (2) proteínas da membrana peritrófica; (3) envolvidas com proteção; (4) transportadores; (5) receptores; (6) proteínas da maquinaria secretória; (7) proteínas de citoesqueleto; (8) com função desconhecida nesse local. Em ambas as preparações existem uma predominância de sequências de enzimas digestivas. Nas membranas microvilares a maioria das sequências são aminopeptidases, enquanto nas vesículas microapócrinas a maioria são lipases. Os cDNAs correspondentes as proteínas que poderiam estar envolvidas na maquinaria secretória foram clonados e sequenciados. São elas: fimbrina, cofilina, gelsolina-1 e miosina I. RT-PCRs semi-quantitativas dessas proteínas em diferentes tecidos do inseto (intestino, túbulos de Malpighi, corpo gorduroso e carcaça) mostraram que somente gelsolina-1 está presente exclusivamente no intestino. Os domínios G1-G3 da gelsolina característica do intestino (gelsolina-1) foram expressos e usados para produzir anticorpos em coelhos. Esses anticorpos reconhecem a proteína recombinante e uma proteína presente no epitélio intestinal com massa molecular compatível com a massa predita para gelsolina-1. Foi possível diminuir a expressão de gelsolina-1 utilizando RNA interferente.Lepidoptera anterior midgut presents a microapocrine secretion of digestive enzymes with secretory vesicles migrating inside the microvilli. These vesicles bud from the midgut microvilli as double membrane vesicles and are discharged into the lumen. The aim of this work was to identify the proteins secreted and those involved in the microapocrine secretory machinery in Spodoptera frugiperda larvae. For this, microapocrine vesicles were prepared and used for polyclonal antibody production. This antibody was used to screen a cDNA expression library of S. frugiperda midgut. We also obtained a transcriptome by pyrosequencing a cDNA library derived from transcripts of the midgut. Positive clones from the screening were sequenced, assembled and N-blasted against S. frugiperda sequences obtained by pyrosequencing. This procedure led to the extension of the sequences previously obtained. We also used the sequences generated by pyrosequencing to reanalyze the sequences of microvillar membrane proteins obtained previously by Ferreira et al. (2007). This reanalysis generated 66 predicted proteins that are present in the microvillar membranes. Eighteen were considered to be contaminants from other compartments and 48 associated with the microvillar membranes. Analysing the sequences from microapocrine vesicles we found 50 predicted proteins that should be secreted by microapocrine vesicles. The sequences found in both microvillar membrane and microapocrine vesicles may be classified into 8 groups, according to their function: (1) digestive enzymes; (2) peritrophic membrane proteins; (3) protection; (4) transporters; (5) receptors; (6) secretory machinery; (7) cytoskeleton; (8) with unknown function. In both preparations there is a predominance of sequences of digestive enzymes. In microvillar membranes, there is a remarkable amount of aminopeptidases, while in microapocrine vesicles this is true for lipases. cDNAs coding for proteins that could be involved in the microapocrine secretory machinery were cloned and sequenced. They are: fimbrin, cofilin, gelsolin-1 and myosin I. Using RT-PCR, we showed that mRNAs coding for gelsolin-1 and myosin I are present only in the intestinal tissue. The mRNAs coding for other proteins were found in all tissues. The domains G1-G3 from gelsolin specific from intestinal midgut (gelsolin 1) were expressed and used to raise antibodies in rabbit. These antibodies were able to recognize the recombinant protein and a protein from the midgut epithelium that has a molecular weight similar to the one predicted from gelsolin-1 sequence. We succeed in decreasing the expression of gelsolin-1 by using interfering RN

Effects of croton urucurana extracts and crude resin on Anagasta kuehniella (Lepidoptera: Pyralidae)

Hundreds of plant species have been studied in order to find out the active ingredient responsible for their insecticidal activity against the pests of economic importance. To verify the insecticidal activity in the husk of stem of Croton urucurana Baillon 1864 (Euphorbiaceae) against Anagasta kuehniella Zeller 1879 (Lepidoptera: Pyralidae), the methanolic (EMeOH) extract, dichloromethane fraction (FDM), ethyl acetate fraction (FAE) and crude resin, incorporated into an artificial diet were evaluated. EMeOH (0.5, 1.0 and 2.0%) and crude resin (2.0%) interfered with neither the weight nor the survival of fourth instar larvae and other analyzed parameters. FDM (2.0%) fraction caused mortality of 65%, and the artificial diet containing 2.0, 1.0 and 0.5% FAE caused 100, 55 and 68% mortality respectively when compared with the control, confirming the least efficiency rates of food conversion for FDM(2.0%) and FAE(1.0%). The tryptic analysis performed with the midgut fluid of fourth-instar larvae demonstrated that tryptic and chymiotryptic activities for the larvae fed artificial diet containing EMeOH and crude resin were not different.<br>Atualmente centenas de plantas são investigadas para se conhecer os princípios ativos responsáveis pela atividade inseticida contra as diversas pragas de importância econômica. Com o objetivo de verificar a atividade inseticida das cascas do caule de Croton urucurana Baillon 1864 (Euphorbiaceae) em relação a Anagasta kuehniella Zeller 1879 (Lepidoptera: Pyralidae), avaliou-se o extrato metanólico (EMeOH), fração diclorometano (FDM), a fração acetato de etila (FAE) e a resina in natura, os quais foram adicionados à dieta artificial. O EMeOH (0,5, 1,0 e 20,%) e a resina in natura (2,0%), não interferiram no peso, sobrevivência das larvas de 4ª ínstar, bem como nos demais parâmetros analisados. A fração FDM (2,0%) causou mortalidade de 65%, e a dieta artificial contendo 2,0, 1,0, e 0,5% de FAE causou 100, 55 e 68% de mortalidade respectivamente quando comparado com o controle, constatando-se as menores eficiências de conversão do alimento para a FDM (2,0%) e FAE (1,0%). Através do ensaio tríptico realizado com o fluido do intestino médio das larvas, foi possível verificar que a atividade tríptica e quimiotríptica para as larvas alimentadas com dieta artificial contendo o EMeOH e a resina in natura não apresentaram diferenças significativas

Evaluation of the Adenanthera pavonina seed proteinase inhibitor (ApTI) as a bioinsecticidal tool with potential for the control of Diatraea saccharalis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Diatraea saccharalis, is a major sugarcane pest, causing damage to the stalks of sugarcane plants. In this study, a trypsin inhibitor (ApTI) was purified from Adenanthera pavonina seeds and was tested for its insect growth regulatory effect. ApTI showed a dose-dependent effect on average larval weight and survival. 0.1% ApTI produced approximately 67% and 50% decreases in weight and survival larval, respectively. The results from dietary utilization experiments with D. saccharalis larvae showed a reduction in the efficiency of conversion of ingested food and digested food, and an increase in approximate digestibility and metabolic cost. The level of trypsin was significantly decreased (ca. 55%) in the midgut of larvae reared on a diet containing 0.05% ApTI and the trypsin activity in ApTI-fed larvae demonstrated sensitivity to ApTI. The action of ApTI on the development of D. saccharalis larvae shows that this protein may have great toxic potential. (C) 2011 Elsevier Ltd. All rights reserved.472257263FUNDECT (Fundacao de Apoio ao Desenvolvimento do Ensino, Ciencia e Tecnologia do Estado de Mato Grosso do Sul)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Evaluation of the Adenanthera pavonina seed proteinase inhibitor (ApTI) as a bioinsecticidal tool with potential for the control of Diatraea saccharalis

Diatraea saccharalis, is a major sugarcane pest, causing damage to the stalks of sugarcane plants. In this study, a trypsin inhibitor (ApTI) was purified from Adenanthera pavonina seeds and was tested for its insect growth regulatory effect. ApTI showed a dose-dependent effect on average larval weight and survival. 0.1% ApTI produced approximately 67% and 50% decreases in weight and survival larval, respectively. The results from dietary utilization experiments with D. saccharalis larvae showed a reduction in the efficiency of conversion of ingested food and digested food, and an increase in approximate digestibility and metabolic cost. The level of trypsin was significantly decreased (ca. 55%) in the midgut of larvae reared on a diet containing 0.05% ApTI and the trypsin activity in ApTI-fed larvae demonstrated sensitivity to ApTI. The action of ApTI on the development of D. saccharalis larvae shows that this protein may have great toxic potential. (C) 2011 Elsevier Ltd. All rights reserved.FUNDECT (Fundacao de Apoio ao Desenvolvimento do Ensino, Ciencia e Tecnologia do Estado de Mato Grosso do Sul)FUNDECT (Fundacao de Apoio ao Desenvolvimento do Ensino, Ciencia e Tecnologia do Estado de Mato Grosso do Sul)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico