Purificação, caracterização e análise da atividade bioinseticida, de um inibidor de tripsina em sementes de catanduva (Piptadenia moniliformis)

One Kunitz-type trypsin inhibitors (PmTI) was purified from Piptadenia moniliformis seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose, DEAE cellulose (ion exchange) and Superose 12 (molecular exclusion) column FPLC/AKTA. The inhibitor has Mr of 25 kDa by SDS-PAGE and chromatography molecular exclusion. The N-terminal sequence of this inhibitor showed high homology with other family Kunitz inhibitors. This also stable variations in temperature and pH and showed a small decrease in its activity when incubated with DDT in the concentration of 100mM for 120 minutes. The inhibition of trypsin by PmTI was competitive, with Ki of 1.57 x10-11 M. The activity of trypsin was effectively inhibited by percentage of inhibition of 100%, among enzymes tested, was not detected inhibition for the bromelain, was weak inhibitor of pancreatic elastase (3.17% of inhibition) and inhibited by 76.42% elastase of neutrophils, and inhibited in a moderate, chymotrypsin and papain with percentage of inhibition of 42.96% and 23.10% respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. Several degrees of inhibition were found. For Anthonomus grandis and Ceratitis capitata the inhibition was 89.93% and 70.52%, respectively, and the enzymes of Zabrotes subfasciatus and Callosobruchus maculatus were inhibited by 5.96% and 9.41%, respectively, and the enzymes of Plodia. interpunctella and Castnia licus were inhibited by 59.94% and 23.67, respectively. In vivo assays, was observed reduction in the development of larvae in 4rd instar of C. capitata, when PmTI was added to the artificial diet, getting WD50 and LD50 of 0.30% and 0.33%, respectively. These results suggest that this inhibitor could be a strong candidate to plant management programs cross transgenic

Vicilina de sementes da leguminosa selvagem Anadenanthera macrocarpa (AmV): purificação, caracterização, efeito deletério e mecanismo de ação para bruquídeo callosobruchus maculatus

Grains and legume seeds are foods that form the basis of the diets of many cultures around the world, winch contritbute to the daily nutrient requirements of humans. Vicilins (7S globulin) are storage proteins found in legume seeds, and may have an additional function constitutive defense of the embryo against pests and pathogens. In this work the vicilin from Anadenanthera macrocarpa - AmV (red-angico), was purified and partially characterized, its effect on development and larval survival and adult emergence of Callosobruchus maculatus was evaluated by determination of LD50, WD50 and ED50 in system bioassay. Purification of vicilin was initiated by the chitin affinity chromatography and then gel filtration (Superdex 75 Tricorn 10x300 mm) FPLC system followed by reverse phase chromatography (C8 phenomenex) on HPLC system. Bioassays WD50 and LD50 for larvae were 0.32% and 0.33% (w:w) respectively, since the ED50 for adults was 0.096%. The probable mechanism of action was evaluated by testing digestibility of AmV in vitro, and observed for the involvement of two fragments vicilins immunoreactive against polyclonal Anti-vicilin from Erythrina velutina (Anti-EvV) about of 22 and 13 kDa chitin binding. The AmV in its native form has been recognized by the anti-EvV, indicating that there is a conserved region in the vicilin and is probably corresponding to the chitin binding domains. These results point to a new vicilin chitin binding that can subsequently be used as a possible biopesticide protein source, in order to control insect pest C. maculatus and confirm literature findings that demonstrate vicilin in the presence of different kinds of ligands to conserved regions chitin not yet characterized

Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Atividade antiplasmódica e toxicológica de plantas medicinais usadas popularmente no Brasil : uma abordagem etnobotânica

Malaria is a disease of global distribution, recognized by governments around the world as a serious public health problem, affecting more than 109 countries and territories and endangering more than 3.3 billion people. The economic costs of this disease are also relevant: the African continent itself has malaria-related costs of about $ 12 billion annually. Nowadays, in addition to chloroquine, Plasmodium falciparum is resistant to many drugs used in the treatment of malaria, such as amodiaquine, mefloquine, quinine and sulfadoxine-pyrimethamine; resistance of Plasmodium vivax to treatments, although less studied, is also reported. Nature, in general, is responsible for the production of most known organic substances, and the plant kingdom is responsible for the most of the chemical diversity known and reported in the literature. Most medicinal plants commercialized in Brazil, however, are of exotic origin, which makes the search for endemic medicinal plants, besides a patent necessity, a fascinating subject of academic research and development. This study aimed to: (i) verify the antimalarial activity of ethanolic and hydroalcoholic extracts of Boerhavia paniculata Rich. And acetonic extract of Clethra scabra Pers. in Swiss albino mice infected by Plasmodium berghei NK65, (ii) observe possible combined effects between the course of infection by P. berghei NK65 and administration of these extracts in Swiss albino mice, and (iii) conduct a preliminary study of the acute toxicity of these extracts in Swiss albino mice. All extracts notable pharmacological activities - with parasite infections inhibitions ranging from 22% to 54%.These characteristics suggest that the activities are relevant, although comparatively lower than the activity displayed by the positive control group (always above 90%). The general framework of survival analysis demonstrates an overall reduction in survival times for all groups. Necroscopy has not pointed no change in color, shape, size and/or consistency in the evaluated organs - the only exception was the livers of rats submitted to treatment to hydroalcoholic extracts: these organs have been presented in a slightly congestive aspect with mass increasing roughly 28% higher than the other two groups and a p-value of 0.0365. The 250 mg/Kg ethanolic group has been pointed out by the Dunn s post test, as the only class with simultaneous inequalities (p<0.05) between positive and negative control groups. The extracts, notably ethanol extract, have, in fact, a vestigial antimalarial activity, although well below from the ones perceived to chloroquine-treated groups; nevertheless, the survival times of the animals fed with the extracts do not rise by presence of such therapy. Both the toxicopharmacological studies of the synergism between the clinical course of malaria and administration of extracts and the isolated evaluation of toxicity allow us to affirm the absence of toxicity of the extracts at the level of CNS and ANS, as well as their non-influence on food and water consumption patterns, until dosages of 500 mg/Kg. Necroscopic analysis leads us to deduct a possible hepatotoxic effect of hydroalcoholic extract at dosages of 500 mg/Kg, and an innocuous tissue activity of the ethanol extract, in the same dosage. We propose a continuation of the studies of these extracts, with protocol modifications capable of addressing more clearly and objectively their pharmacological and toxicological aspects

Thiamethoxam on the histological characteristics of sugarcane young roots

Thiamethoxam is a systemic insecticide from the neonicotinoid group, nitroguanidin family which affects the nicotinic receptor acetyl choline in the insect membrane, wounding the nervous system and causing the death of the insect. It was used with success in the control of initial pests of several crops. It was considered that thiamethoxam has a bioactivator effect, because in the absence of insects promoted increase in vigor, development and productivity of crops. This work was carried out to verify if thiamethoxam causes histological changes in sugarcane roots. In this work, it was used optical microscopy, images arrest, tissue biometrics and statistical analysis, in young roots of sugarcane RB 83 5486 after the treatments with different thiamethoxam concentrations. It was determined changes in histological structure of tissues 7, 14, 21 and 28 days after the treatments, establishing its effects on root plant anatomy. It was verified that thiamethoxam increased root cortex width, increasing the vascular cylinder and the metaxylem vessel elements number in the vascular tissue until 21 days after application.

Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization and selection of Bacillus thuringiensis isolates effective against Sitophilus oryzae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação dos parâmetros genéticos em progênies de meio-irmãos de uma sub-população de milho composto Flint para resistência à lagarta-da-espiga Helicoverpa zea (Bod.)

Foram estudadas 100 progênies de meio-irmãos de uma sub-população de milho (Zea mays L.) Composto Flint com o objetivo de avaliar a resistência de genótipos à lagarta-da-espiga Helicoverpa zea (Bod.). Foram obtidos os valores de danos médios da lagarta-da-espiga de 1,14 cm de comprimento na espiga determinado pela escala de Widstrom e coeficiente de variação experimental (CVE) de 23,4%. Dos parâmetros genéticos avaliados, a estimativa de herdabilidade (h²) foi de 6%, variância genética (VG) de 0,0015 cm² e variância fenotípica (VF) de 0,025 cm² para danos de H. zea. No entanto, o comprimento da ponta da bráctea e compactação da bráctea alcançaram resultados de herdabilidade de 75% e 72% respectivamente. Essa sub-população de milho apresenta variabilidade genética suficiente para utilização em programas de melhoramento, sendo que a resistência à lagarta-da-espiga pode ser obtida através da melhoria dos caracteres morfológicos diretamente relacionados à praga, como a compactação e comprimento da bráctea.

Planos de amostragem seqüenciais para Orthezia praelonga Douglas (Hemiptera: Sternorrhyncha, Ortheziidae) na cultura de citros

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Amostragem seqüencial (presença-ausência) para Bemisia tabaci (Genn.) biótipo B (Hemiptera: Aleyrodidae) em feijoeiro (Phaseolus vulgaris L.)

A amostragem seqüencial (presença-ausência) vem sendo utilizada no manejo integrado de pragas pela rapidez e eficiência, principalmente, para pragas que são difíceis de serem quantificadas. Para o manejo de Bemisia tabaci (Genn.) biótipo B foi desenvolvido um plano de amostragem seqüencial, com base na presença ou ausência da praga em plantas de feijoeiro (Phaseolus vulgaris L.), independente do seu número. Os experimentos foram conduzidos nas épocas de semeadura das águas (2000/01) e da seca (2002), em Jaboticabal - SP, utilizando-se área de 1 ha, subdividida em 100 parcelas iguais de 100 m² (10 x 10 m). em cada parcela foram avaliadas 10 plantas ao acaso, considerando-se somente se a mosca-branca estava ou não presente. O nível de dano econômico adotado foi de 10% de infestação. A partir dos dados analisados, foram obtidas duas retas: uma superior (S1= 2,7095 + 0,1452n), a partir da qual recomenda-se o controle; e outra inferior (S0= -2,7095 + 0,1452n), até a qual o controle não é recomendado. Pelos resultados verifica-se que a amostragem seqüencial é eficiente na indicação ou não do controle da B. tabaci biótipo B na cultura do feijão.

Amostragem seqüencial com base na lei de Taylor para levantamento de Spodoptera frugiperda na cultura do milho

A lagarta do cartucho do milho é uma das principais pragas do milho nas Américas, podendo ocorrer durante todos os estágios de crescimento da cultura, causando perdas de 15 a 37% na produção. Neste trabalho estudou-se a distribuição espacial dessa praga no campo, com a finalidade de desenvolver um sistema de amostragem para estimar sua densidade populacional. Na área cultivada com milho foram selecionados 3 campos experimentais de 40 m x 250 m cada, divididos em 100 parcelas cada. Foram contados o número de lagartas pequenas e grandes de S. frugiperda nas folhas e no cartucho das plantas, em 10 plantas ao acaso por parcela, num total de 1000 plantas por campo, em 5, 5 e 4 datas de amostragem, respectivamente. Lagartas menores que 1 cm eram consideradas pequenas e lagartas maiores que 1 cm, grandes. Foram estimados os parâmetros da lei de Taylor, sendo as estimativas dos parâmetros das regressões altamente significativas para todas as categorias larvais. O parâmetro b da lei de Taylor para lagarta pequena foi igual a 1,57, indicando que a forma de distribuição dessa categoria larval é agregada. Para lagartas grandes o parâmetro b foi igual a 0,79, o que conduz a um tipo de distribuição tendendo para aleatoriedade. Para o número total de lagartas os resultados são equivalentes aos obtidos para lagartas pequenas. Foi desenvolvido um plano de amostragem seqüencial com base nos parâmetros da lei de Taylor.

Spatial distribution of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) in citrus orchards

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Distribuição de probabilidade de ocorrência de Orthezia praelonga Douglas (Hemiptera: Sternorrhyncha: Ortheziidae) na cultura de citros

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Distribuição espacial de Dilobopterus costalimai young (Hemiptera: Cicadellidae) em citros na região de Taquaritinga, SP

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Distribuição espacial de Toxoptera citricida (Kirkaldy) (Hemiptera: Aphididae) na cultura de citros

O estudo da distribuição espacial de pragas é fundamental para elaboração de planos de amostragem para o uso do manejo integrado de pragas. Para o afídeo Toxoptera citricida (Kirkaldy), estudou-se a distribuição espacial em talhões de pomares de citros comerciais de laranja-doce [Citrus sinensis (L.) Osbeck] da variedade Pêra, com 5; 9 e 15 anos de idade, durante o período de setembro de 2004 a abril de 2005. Foram realizadas 14 amostragens de número de T. citricida em intervalos aproximados de 15 dias entre as mesmas, utilizando-se de armadilhas adesivas de cor amarela (0,11 x 0,11 m) fixadas à planta, a 1,5 m de altura aproximadamente. As armadilhas foram distribuídas na área, a cada cinco plantas na linha, em linhas alternadas, totalizando 137 armadilhas no talhão com 5 anos, 140 no talhão com 9 anos e 80 no talhão com 15 anos. Os índices de dispersão utilizados foram: razão variância média (I), índice de Morisita (Idelta), coeficiente de Green (Cx) e expoente k da distribuição Binomial Negativa. O índice que melhor representou a agregação do pulgão foi o expoente k da distribuição Binomial Negativa, e a distribuição binomial negativa foi o modelo que melhor se ajustou aos dados. Através destas análises, verificou-se que a maioria das amostragens apresentou uma distribuição agregada da população de T. citricida.