RESUMO: Bacillus thuringiensis subsp. israelensis vem sendo empregada no controle do díptero Aedes aegypti, vetor do vírus causador de doenças como dengue, febre Chikungunya e Zika, que se tornou um dos grandes problemas de saúde pública no Brasil e em outros países de clima tropical, que favorece a proliferação e o desenvolvimento do transmissor. Em virtude do potencial de B. thuringiensis no controle de dípteros, a proposta deste trabalho foi testar as proteínas Cry2Aa e Cry2Ab da linhagem de Bacillus thuringiensis subsp. thuringiensis T01-328 no controle de A. aegypti, em comparação à linhagem díptero específica B. thuringiensis subsp. israelensis. Para tanto, foram realizados bioensaios com larvas de A. aegypti com o esporo-cristal de ambas as linhagens, bem como com as proteínas Cry2Aa e Cry2Ab com expressão heteróloga em Escherichia coli. A linhagem B. thuringiensis thuringiensis T01-328 apresentou atividade inseticida contra as larvas, porém foi menos tóxica que a B. thuringiensis subsp. israelensis. As proteínas Cry2Aa e Cry2Ab expressas de forma heteróloga foram eficazes no controle de A. aegypti. Os resultados obtidos sugerem as proteínas Cry2Aa e Cry2Ab da linhagem B. thuringiensis thuringiensis T01-328 como alternativas para contribuir no controle do A. aegypti.

ABSTRACT: Bacillus thuringiensis subsp. israelensis has been used to control the Aedes aegypti (Diptera: Culicidae) mosquito larvae, the vector of virus diseases such as dengue, Chikungunya and Zika fever, which have become a major public health problem in Brazil and other tropical countries since the climate favors the proliferation and development of the transmitting vector. Because B. thuringiensis has shown potential for controlling insects of the Diptera order, this work aimed at testing the Bacillus thuringiensis subsp. thuringiensis strain T01-328 and its proteins Cry2Aa and Cry2Ab for control A. aegypti and at comparing the results to the B. thuringiensis subsp. israelensis specific dipteran strain. To this end, bioassays using spore-crystal of both strains, and Cry2Aa and Cry2Ab proteins from the heterologous expression in Escherichia coli, were performed against A. aegypti larvae. The results showed that the B. thuringiensis thuringiensis T01-328 has insecticidal activity against the larvae, but it is less toxic than B. thuringiensis subsp. israelensis. Cry2Aa and Cry2Ab proteins expressed heterologously were effective for controlling A. aegypti larvae. Therefore, the results indicate that the Cry2Aa and Cry2Ab proteins of the B. thuringiensis thuringiensis T01-328 can be used as an alternative to assist in the control of A. aegypti.

Resumo: O objetivo deste trabalho foi avaliar a toxicidade e a capacidade de ligação das proteínas Cry1Aa, Cry1Ab, Cry1Ac e Cry1Ca, de Bacillus thuringiensis, a receptores intestinais de Helicoverpa armigera. Realizou-se análise de ligação das proteínas ativadas às vesículas de membrana da microvilosidade apical (VMMA) do intestino médio deH. armigera, além de ensaios de competição heteróloga para avaliar sua capacidade de ligação. Cry1Ac destacou-se como a proteína mais tóxica, seguida por Cry1Ab e Cry1Aa. A proteína Cry1Ca não foi tóxica às lagartas e, portanto, não foi possível determinar os seus parâmetros de toxicidade CL50 e CL90. As proteínas Cry1Aa, Cry1Ab e Cry1Ac são capazes de se ligar a um mesmo receptor nas membranas intestinais, o que aumenta o risco do desenvolvimento de resistência cruzada. Portanto, a utilização conjunta dessas proteínas deve ser evitada.

Abstract: The objective of this work was to evaluate the toxicity and the binding capacity of the Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ca proteins, from Bacillus thuringiensis, to Helicoverpa armigera intestine receptors. Binding analysis of the activated proteins to the brush-border membrane vesicles (BBMV) in the midgut of H.armigera, besides heterologous competition assays to evaluate their binding capacity, was performed. Cry1Ac stood out as the most toxic protein, followed by Cry1Ab and Cry1Aa. The Cry1Ca protein had no toxicity to the caterpillars and, therefore, it was not possible to evaluate its LC50 and LC90 toxicity parameters. The Cry1Aa, Cry1Ab, and Cry1Ac proteins are able to bind themselves to the same receptor in the midgut membrane, which increases the risk of developing cross-resistance. Therefore, the use of these proteins together should be avoided.

Resumo:O objetivo deste trabalho foi avaliar a toxicidade de novas proteínas Vip3Aa e sua capacidade de ligação a vesículas de membrana da microvilosidade apical (VMMA) do intestino de lagartas neonatas de Spodoptera frugiperda, Anticarsia gemmatalise Heliothis virescens. Proteínas expressas pelos genes vip3Aa42 e vip3Aa43 mostraram-se tóxicas a S. frugiperda (CL50 de 78,2 e 113 ng cm-2, respectivamente) e A. gemmatalis(CL50 de 239,2 e 57,5 ng cm-2, respectivamente), e pouco tóxicas a H. virescens (CL50>5.000 ng cm-2). Os ensaios de ligação às VMMA mostraram que as proteínas unem-se de forma efetiva aos receptores nas vesículas das espécies avaliadas, mas essa capacidade de ligação somente é efetiva na ativação da toxicidade para as populações avaliadas de S. frugiperdae A. gemmatalis.

Abstract:The objective of this work was to evaluate the toxicity of new Vip3Aa proteins and their binding capacity to brush-border membrane vesicles (BBMV) in the intestine of Spodoptera frugiperda, Anticarsia gemmatalis, and Heliothis virescens neonate larvae. The proteins expressed by the genes vip3Aa42 and vip3Aa43 showed toxicity to S. frugiperda (LC50 of 78.2 and 113 ng cm-2, respectively) and A. gemmatalis(LC50 of 239.2 and 57.5 ng cm-2, respectively), but they showed low toxicity to H. virescens(LC50>5,000 ng cm-2). BBMV binding assays showed that the proteins bind effectively to the receptors on vesicles of the evaluated species, but this binding capacity is only effective on the activation of toxicity to the evaluated populations of S. frugiperdaand A. gemmatalis.

O objetivo deste trabalho foi avaliar a suscetibilidade das lagartas Anticarsia gemmatalis (Lepidoptera: Erebidae) e Chrysodeixis includens (Lepidoptera: Noctuidae) às proteínas Cry1 e Vip3A, bem como determinar se há a interação entre essas proteínas no controle das duas espécies. Bioensaios com as proteínas isoladas e em combinações foram realizados, e as concentrações letais CL50 e CL90 foram estimadas para cada condição. As proteínas Cry1Aa, Cry1Ac e Vip3Af foram as mais efetivas no controle de A. gemmatalis, enquanto Cry1Ac, Vip3Aa e Vip3Af foram mais efetivas no de C. includens. As proteínas Cry1Ac e Cry1Ca causaram maior inibição do desenvolvimento das larvas sobreviventes à CL50, em ambas as espécies. Combinações entre Vip3A e Cry1 apresentam efeito sinérgico no controle das espécies e a combinação Vip3Aa+Cry1Ea destaca-se no controle de A. gemmatalis e C. includens. Essas proteínas combinadas são promissoras na construção de plantas piramidadas, para o controle simultâneo das pragas.

The objective of this work was to evaluate the susceptibility of Anticarsia gemmatalis (Lepidoptera: Erebidae) and Chrysodeixis includens (Lepidoptera: Noctuidae) caterpillars to Cry1 and Vip3A proteins, as well as to determine if there is any interaction between these proteins on the control of the two species. Bioassays with both isolated and combined proteins were carried out, and lethal concentrations LC50 and LC90 were estimated for each condition. Cry1Aa, Cry1Ac, and Vip3Af were the more effective proteins for the control of A. gemmatalis, while Cry1Ac, Vip3Aa, and Vip3Af were more effective for the control of C. includens. Cry1Ac and Cry1Ca proteins caused the highest inhibition to the development of larvae that survived the LC50 dose in both species. Different combinations of Vip3A and Cry1 have synergistic effect in the control of both species, and the combination Vip3Aa + Cry1Ea showed an outstanding control of A. gemmatalis and C. includens. These proteins are promising for building pyramided plants for the simultaneous control of the pests.

O objetivo deste trabalho foi caracterizar o gene vip3A de Bacillus thuringiensis e verificar a toxicidade da proteína Vip3Aa50 a larvas da lagarta-do-cartucho (Spodoptera frugiperda) e da lagarta-da-soja (Anticarsia gemmatalis). O gene vip3A foi amplificado por PCR, com iniciadores específicos, e gerou um fragmento de 2.370 pb. Esse fragmento foi clonado em vetor pGEM-T Easy e, em seguida, sequenciado, subclonado em vetor de expressão pET-28a (+) e inserido em células de Escherichia coli BL21 (DE3). A expressão da proteína Vip3Aa50 foi induzida por isopropil-β-D-1-tiogalactopiranosídeo (IPTG), visualizada em SDS-PAGE e detectada por "Western blot". Os ensaios de toxicidade revelaram alta atividade da proteína Vip3Aa50 contra as larvas neonatas da lagarta-da-soja e da lagarta-do-cartucho, com CL50 de 20,3 e 79,6 ng cm-2, respectivamente. O gene vip3Aa50 é um novo gene da classe vip3A.

The objective of this work was to characterize the vip3A gene of Bacillus thuringiensis and to evaluate the toxicity of Vip3Aa50 protein to the fall armyworm (Spodoptera frugiperda) and velvetbean caterpillar (Anticarsia gemmatalis) larvae. The gene vip3A was amplified by specific PCR primers, generating a 2,370-bp fragment. This fragment was cloned into the pGEM-T Easy vector, and then it was sequenced, subcloned into the pET-28a (+)'s expression vector, and inserted into Escherichia coli BL21 (DE3) cells. The Vip3Aa50 protein expression was induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG), visualized in SDS-PAGE, and detected by Western blot. The toxicity bioassay showed a high activity of Vip3Aa50 protein against both velvetbean and fall armyworm neonate larvae, with LC50 at 20.3 and 79.6 ng cm-2 respectively. The vip3Aa50 gene, is a new gene of vip3A class.

O flamboyanzinho (Caesalpinia pulcherrima, Fabaceae) é muito utilizado como cerca-viva e na arborização de ruas, parques e jardins. De florescimento exuberante, suas flores podem apresentar coloração rosa, laranja ou amarela, conforme a variedade. Como características florais são essenciais para a definição do valor comercial de espécies ornamentais, o objetivo principal do trabalho foi verificar a existência de polimorfismo entre plantas de C. pulcherrima, que produzem flores de diferentes colorações, por marcadores moleculares RAPD. Foram escolhidas aleatoriamente 30 plantas adultas, cultivadas no município de Jaboticabal, Estado de São Paulo, para a retirada de amostras foliares para a extração de DNA. Dentre essas matrizes, 20 possuem flores alaranjadas, oito, flores amarelas, e apenas duas produzem flores de coloração rosa. Dos 140 primers de RAPD avaliados, 94 foram capazes de ampliar fragmentos definidos, gerando 246 bandas, das quais se observou 100% de bandas monomórficas, indicando que nenhum dos primers utilizados detectou polimorfismo entre os tratamentos. Concluiu-se que a técnica para detecção de polimorfismo por marcadores RAPD não foi eficiente, e que existe a necessidade de se testarem marcadores moleculares mais específicos. Além disso, a característica morfológica "cor de flor" é, possivelmente, controlada por vários genes ou pela combinação deles.

The species Caesalpinia pulcherrima, which belongs to the Fabaceae family, is frequently used as a hedge or street tree in urban landscapes, parks and gardens. It flowers profusely, and the flowers, depending on the variety, may be pink, orange or yellow. As flower characteristics are essential for determining the commercial value of ornamental species, the main objective of this work was to identify polymorphisms in C. pulcherrima plants that produce flowers of different colors using RAPD molecular markers. For this study, 30 adult plants were randomly chosen on streets located in Jaboticabal city, São Paulo State, Brazil, and leaf samples were collected for DNA extraction. Among these trees, 20 have orange flowers, eight have yellow flowers and only two produce pink flowers. From the 140 tested RAPD primers, 94 primers amplified defined fragments that resulted in 246 bands, which were 100% monomorphic. Thus, a polymorphism was not detected by any of the primers tested. It was concluded that the RAPD technique is not an efficient method for detecting polymorphisms and that more specific molecular markers should be tested. Additionally, the morphological characteristic "flower color" may be controlled by several genes or by the association of them.

O objetivo deste trabalho foi identificar e caracterizar os genes cry3, vip1, vip2 e vip1/vip2 em uma coleção de 1.078 isolados de Bacillus thuringiensis potencialmente tóxicos para larvas de coleópteros. Foram utilizados pares de oligonucleotídeos iniciadores gerais obtidos a partir de regiões conservadas dos genes e do alinhamento de sequências consenso. Posteriormente, os isolados positivos foram caracterizados por meio da técnica de PCR‑RFLP, tendo-se utilizado enzimas de restrição específicas, para identificar novas subclasses de genes nos isolados. Cento e cinquenta e um isolados foram positivos para os genes avaliados, com maior frequência para o gene vip1/vip2 (139 isolados). Pela técnica de PCR‑RFLP, foram observados 14 perfis polimórficos, o que indica a presença de diferentes alelos e, consequentemente, de distintas subclasses desses genes.

The objective of this work was to identify and characterize cry3, vip1, vip2 and vip1/vip2 genes in a collection of 1,078 Bacillus thuringiensis isolates potentially toxic against Coleoptera larvae. Pairs of primers derived from conserved regions of genes and from sequence alignment consensus were used. Subsequently, positive isolates were characterized by PCR‑RFLP, using specific restriction enzymes to identify new subclasses of genes in the isolates. One hundred and fifty‑one isolates were positive for the evaluated genes, with higher frequency for the vip1/vip2 gene (139 isolates). By PCR‑RFLP, 14 polymorphic profiles were observed, indicating the presence of different alleles, and, therefore, of distinct subclasses of these genes.

Aedes aegypti (L.), the main vector of dengue fever in Brazil, has been controlled with the use of massive chemical products, contributing to the development of resistance and decreasing the insect control efficiency. The control of dipterans with bioinsecticides based on Bacillus thuringiensis has been satisfactory, due to the production of insecticidal proteins denominated Cry (crystal), Cyt (cytolytic) toxins and Chi (chitinase), and to the synergistic effects among them. The present work aimed to select B. thuringiensis isolates efficient against A. aegypti larvae. A bacterial collection containing 1,073 isolates of B. thuringiensis, obtained from different locations of Brazilian territory, had the DNA isolated and submitted to PCR amplifications using specific primers for cry4Aa, cry4Ba, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa and chi genes. For the LC50 and LC90 determination, the entomopathogenic isolates were evaluated by selective and quantitative bioassays. Only 45 isolates (4.2%) presented amplicons for the cry and cyt genes. The chi gene sequence was detected in 25 (54.3%) of those isolates. From the 45 isolates submitted to the selective bioassays, 13 caused 100% mortality of A. aegypti larvae. The identification of cry, cyt and chi genes of B. thuringiensis and the toxicity analysis on A. aegypti led to the selection of a set of isolates that have the potential to be used in the formulation of new bioinsecticides.

Sabendo-se da influência das mutações no gene TP53 no desenvolvimento das neoplasias e da discrepância entre os resultados obtidos pelas técnicas de sequenciamento e imunoistoquímica, esta pesquisa teve como objetivo relacionar a sequência do TP53 com a imunorreatividade da p53. Foram obtidas amostras de linfoma de 12 cães. O diagnóstico histopatológico foi determinado pela classificação de Kiel. O imunofenótipo e a imunomarcação da p53 foram determinados por imunoistoquímica. Para reação com a p53, utilizou-se anticorpo policlonal anti-p53 (CM1) na diluição de 1:500. A região do gene TP53 compreendida entre os exons quatro e nove foi amplificada por PCR e submetida ao sequenciamento. Apesar dos resultados obtidos pela imunoistoquímica, nenhuma mutação foi encontrada nas sequências analisadas. Conclui-se que a imunorreatividade da p53 pela imunoistoquímica não pode ser atribuída à presença de mutações no domínio central do gene TP53.

TP53 mutations are usually involved in cancer, but sequencing and immunohistochemistry results are often controversial. Thus, the aim of this study was to associate TP53 sequence with p53 immunostaining in dogs with lymphoma. Tumor samples were collected from 12 dogs with lymphoma and were included in this study. Histopathological diagnosis was performed according to Kiel classification. Immunohistochemistry was performed to identify immunophenotype as well as p53 expression. Polyclonal antibody anti-p53 (CM1) was used at a 1:500 dilution. The region that encompasses exons 4-9 was amplified by f PCR reactions and sequencing was then performed. Nevertheless, gene mutations were not observed in any sequence. In conclusion, immunoreactivity of p53 by means of immunohistochemistry should not be indicator of presence of mutations in the core domain of TP53 gene.

As espécies de Digitaria são plantas daninhas nas lavouras de cana-de-açúcar no Brasil e estão sendo controladas com herbicidas, porém há alguns relatos de falha no controle, notavelmente ao grupo das triazinas. Técnicas moleculares são recomendadas para análise da variabilidade genética em plantas daninhas, como o RAPD (DNA Polimórfico Amplificado ao Acaso) e PCR-RFLP (Reação da Polimerase em Cadeia - Polimorfismo no Comprimento de Fragmentos de Restrição) e, aliadas ao sequenciamento, permitem a localização de genes de resistência, bem como possíveis mutações relacionadas ao surgimento de indivíduos resistentes em algumas espécies. Com isso, o objetivo deste trabalho foi caracterizar dez acessos de Digitaria spp. por meio de marcadores RAPD e PCR-RFLP, sequenciar uma região conservada do gene psbA e analisar a resposta destes à ametrina. Pela análise molecular houve elevada similaridade genética entre os acessos, pois todos apresentaram perfis genéticos semelhantes e foram suscetíveis à ametrina.

Digitaria species are sugar cane crop weeds in Brazil and are being controlled with herbicides, although there are some reports of control failure, notably to the triazine group. Molecular techniques are recommended to analyze the genetic variability in weeds. RAPD (Random Amplified Polymorphic DNA), PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) and, in combination with sequencing, allow the localization of resistance genes, as well as possible mutations related to the onset of resistant individuals in some species. Thus, the objective of this work was to characterize ten accessions of Digitaria spp. by RAPD and PCR-RFLP markers, to sequence a conserved region of the psbA gene and evaluate the accessions response to ametryn. As showed by molecular analysis there was high genetic similarity among the accessions, all of them presented similar genetics profiles and were susceptible to ametryn.

A jabuticabeira é considerada uma das fruteiras mais típicas do Brasil. Entretanto, há poucos estudos sobre esta planta na literatura, e mesmo sua classificação botânica é muito controvertida. Este trabalho faz comparações entre as espécies de jabuticabeiras, usando as técnicas de marcadores morfológicos (organografia) e moleculares RAPD. As características morfológicas das plantas, usadas como marcadores morfológicos, foram comparadas com espécimes presentes nos herbários dos Estados de São Paulo e Minas Gerais e com as descrições obtidas em revisão de literatura especializada. As diferenças moleculares entre as espécies foram determinadas por meio do uso de marcadores RAPD. O experimento foi realizado nas cidades de Piracicaba, Jaboticabal e Ituverava do Estado de São Paulo, Brasil. Diferenças morfológicas e moleculares entre as plantas estudadas foram identificadas, e quatro grupos distintos de espécies foram definidos: Myrciaria cauliflora (Mart.) O. Berg, M. coronata Mattos, M. jaboticaba (Vell.) O. Berg. e M. phytrantha (Kiaersk.) Mattos. A técnica de marcadores moleculares, aliada à técnica de marcadores morfológicos, mostrou ser uma ferramenta importante na identificação de espécies de jabuticabeiras.

The jabuticaba tree is considered one of the most typical Brazilian fruit trees. However, few studies of this plant are found in the literature and even its botanical classification is very controversial. The present research reports some comparisons between jabuticaba species, using morphologic (organography) and molecular markers techniques. The morphologic characteristics of the plant used as markers were compared with specimens of the herbaria from São Paulo and Minas Gerais States and with the descriptions reported in the literature. Molecular differences between the species were identified by the use of RAPD markers. The experiment was made in Piracicaba, Jaboticabal and Ituverava municipal districts in São Paulo State, Brazil. Morphologic and molecular differences between the studied plants were identified and four groups of species were defined: Myrciaria cauliflora (Mart.) O. Berg, M. coronata Mattos, M. jaboticaba (Vell.) O. Berg. and Myrciaria phytrantha (Kiaersk.) Mattos. Both molecular and morphologic markers techniques showed to be important tools for the identification of jabuticaba tree species.

Caseins comprise make up about 80% of the total protein content of milk and present polymorphism with changes in the amino acid sequence. Within this abundance of proteins, kappa-casein is noteworthy, since it has been associated with differences in milk yield, composition and processing. The objective of this study was to observe the existence of polymorphism in the kappa-casein gene in female buffaloes. For this purpose, blood samples from 115 female buffaloes, collected with vacutainer by needle punctionure of the jugular vein, were used. for genomic DNA extraction was done from blood samples. The PCR-RFLP and SSCP techniques demonstrated that the studied animals were monomorphic for the kappa-casein gene. Only allele B was observed in these animals, which was present in homozygosis. Therefore, it was not possible to quantify the gene action on milk yield and its constituents. The monomorphism observed in the population studied would allow the development of a method to identify mixtures of cow and buffalo milk in mozzarella cheese production, especially because, in cattle, the kappa-casein gene is polymorphic.