Abstract The zoanthid Palythoa caribaeorum secretes a large quantity of mucus that acts as a substrate for microbial communities. Culturable bacteria associated with P. caribaeorum mucus in northeastern Brazil were evaluated through 16S rRNA gene sequences. Proteobacteria was the dominant group, followed by Actinobacteria and Firmicutes. Vibrio was the most common genus, although other groups with the ability to produce biosurfactants and compounds with antimicrobial activity were also present. The studies of culturable marine bacteria may contribute to the understanding of the associated microbial community, opening new opportunities to explore biotechnological potential of microbiota.

RESUMO: O controle de baratas realizado através da aplicação de inseticidas químicos exerce alta pressão seletiva sobre as populações e introduz substâncias sintéticas no ambiente, motivando a procura por outros métodos de controle, como os fungos entomopatogênicos. Os objetivos deste trabalho foram investigar a patogenicidade do isolado JAB 42 de Aspergillus westerdijkiae a fêmeas e ootecas de Periplaneta americana e demonstrar o mecanismo de ação sobre ootecas. Suspensões contendo 106 a 108 conídios/mL do isolado foram usadas para infectar fêmeas e ootecas. A ação do fungo foi analisada pela mortalidade e outras variáveis, e por microscopia eletrônica de varredura. O isolado JAB 42 de A. westerdijkiae é patogênico a ootecas de P. americana, tendo baixa capacidade de matar fêmeas. Foi observada a adesão, germinação, penetração e extrusão do fungo sobre ootecas da barata.

ABSTRACT: Cockroach control is performed by the application of chemical insecticides which exert high selective pressure on populations and introduces synthetic substances in the environment, motivating the search for other methods of control such as entomopathogenic fungi. The objectives of this study were to investigate the pathogenicity of the JAB 42 Aspergillus westerdijkiae to females and oothecae of Periplaneta americana and to demonstrate its mechanism of action on oothecae. Suspensions containing 106 to 108 conidia/ml were used to infect females and oothecae. Mortality and other variables such as scanning electron microscopy were used to demonstrate the mechanism of action of the fungus. The isolated JAB 42 A. westerdijkiae is pathogenic to oothecae of P. americana, with low capacity to kill females. Adhesion, germination, penetration and extrusion of the fungus on the cockroach oothecae were observed.

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.

Thirty nine isolates of Flavobacterium columnare from Brazilian fish farms had their carbohydrate composition of EPS evaluated by high efficiency liquid chromatography, using the phenol-sulfuric acid method of EPS. The occurrence of capsules on F. columnare cells was not directly related to biofilm formation, and the predominant monosaccharide is glucose.

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.

Xylella fastidiosa inhabits the plant xylem, a nutrient-poor environment, so that mechanisms to sense and respond to adverse environmental conditions are extremely important for bacterial survival in the plant host. Although the complete genome sequences of different Xylella strains have been determined, little is known about stress responses and gene regulation in these organisms. In this work, a DNA microarray was constructed containing 2,600 ORFs identified in the genome sequencing project of Xylella fastidiosa 9a5c strain, and used to check global gene expression differences in the bacteria when it is infecting a symptomatic and a tolerant citrus tree. Different patterns of expression were found in each variety, suggesting that bacteria are responding differentially according to each plant xylem environment. The global gene expression profile was determined and several genes related to bacterial survival in stressed conditions were found to be differentially expressed between varieties, suggesting the involvement of different strategies for adaptation to the environment. The expression pattern of some genes related to the heat shock response, toxin and detoxification processes, adaptation to atypical conditions, repair systems as well as some regulatory genes are discussed in this paper. DNA microarray proved to be a powerful technique for global transcriptome analyses. This is one of the first studies of Xylella fastidiosa gene expression in vivo which helped to increase insight into stress responses and possible bacterial survival mechanisms in the nutrient-poor environment of xylem vessels.

A bactéria Bacillus thuringiensis Berliner produz um corpo de inclusão paraesporal (cristal) de natureza proteica, formado durante a esporulação, que atua de forma eficiente no controle de insetos-praga de culturas economicamente importantes. Esse cristal é constituído de proteínas Cry, que são codificadas pelos genes cry; um isolado pode ser caracterizado pelo conteúdo de genes cry que apresenta. Visando caracterizar novos isolados no combate de insetos-praga pertencentes às ordens Lepidoptera e Coleoptera, 76 isolados bacterianos foram analisados molecularmente e tiveram seu potencial de controle avaliado por meio de bioensaios com larvas de Spodoptera frugiperda (J.E. Smith), Sphenophorus levis Vaurie e Tenebrio molitor Linnaeus. As análises moleculares indicaram 11 isolados (14,5% da coleção), contendo genes lepidóptero-específicos e 17 (22,37%) com genes coleóptero-específicos. As análises de patogenicidade revelaram dois isolados com alto potencial de controle para lagartas de S. frugiperda, um para larvas de S. levis e seis prejudiciais ao desenvolvimento das larvas de T. molitor. Esses isolados de B. thuringiensis podem ser promissores no controle biológico das referidas pragas.

Bacillus thuringiensis Berliner produces protein crystals that act effectively on the control of pests for several relevant crops. The cry genes codes for these proteins are named Cry proteins. The cry gene profile can potentially set each bacterial isolate as a potential pest control agent. Aiming to analyze new isolates for the controlling of pests belonging to the orders Lepidoptera and Coleoptera, 76 samples were molecularly analyzed and had their potential for control evaluated through bioassays with larvae of Spodoptera frugiperda (J.E. Smith), Sphenophorus levis Vaurie and Tenebrio molitor Linnaeus. Based on molecular analysis data, 11 samples (14.5%) were able to control lepidopteran specific pests due to their cry genes content, and 17 samples (22.37%) exhibited coleopteran specific cry genes. The bioassays results indicated two samples with highly effective against S. frugiperda, one against S. levis and six with high toxicity against T. molitor larvae development. The selected B. thuringiensis isolates may be promising for the biological control of these pests.

Salmonella enterica serovar Typhimurium synthesizes cobalamin (vitamin B12) only during anaerobiosis. Two percent of the S. Typhimurium genome is devoted to the synthesis and uptake of vitamin B12 and to B12-dependent reactions. To understand the requirement for cobalamin synthesis better, we constructed mutants of Salmonella serovars Enteritidis and Pullorum that are double-defective in cobalamin biosynthesis (ΔcobSΔcbiA). We compared the virulence of these mutants to that of their respective wild type strains and found no impairment in their ability to cause disease in chickens. We then assessed B12 production in these mutants and their respective wild type strains, as well as in S. Typhimurium ΔcobSΔcbiA, Salmonella Gallinarum ΔcobSΔcbiA, and their respective wild type strains. None of the mutants was able to produce detectable B12. B12 was detectable in S. Enteritidis, S. Pullorum and S. Typhimurium wild type strains but not in S. Gallinarum. In conclusion, the production of vitamin B12 in vitro differed across the tested Salmonella serotypes and the deletion of the cbiA and cobS genes resulted in different levels of alteration in the host parasite interaction according to Salmonella serotype tested.

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.

O objetivo deste trabalho foi detectar a presença de arqueias no rúmen bovino por meio das sequências gênicas da região conservada 16S rDNA. As amostras foram coletadas de bovinos alimentados com duas dietas experimentais contendo diferentes relações volumoso e concentrado. Para amplificação da região ribossomal 16S rDNA foi feita PCR e a análise das sequências foi realizada pelos programas Phred/Phrap/Consed. As análises do BLAST permitiram identificar no tratamento com 70% de feno, 96 sequências relacionadas à família Methanobacteriaceae, 47 sequências a arqueias não cultiváveis e 60 sequências foram de arqueias desconhecidas e no tratamento com 30% de feno foram 125 sequências relacionadas à família Methanobacteriaceae, 42 sequências a arqueias não cultiváveis e 32 sequências foram de arqueias desconhecidas. A análise das sequências da região 16S rDNA de arqueias do rúmen bovino permitiu detectar maior número de sequências relacionadas com arqueias desconhecidas no tratamento com 70% de feno.

The objective of this research was to detect the presence of archaea in the bovine rumen using genetic sequences from the conserved region of the 16S rDNA. Samples were collected from bovines that were fed two different experimental dietetic ratios of roughage to concentrate. The 16S rDNA region was amplified using PCR and analyzed using the Phred/Phrap/Consed programs. For the treatment with 70% hay diet 96 sequences related to the Methanobacteriaceae family, 47 sequences from non-cultured archaea, and 60 sequences from unknown archaea were identified by the BLAST analysis. For the treatment with 30% hay diet the BLAST analysis identified 125 sequences belonging to the Methanobacteriaceae fa-mily 42 sequences from non-cultured archaea, and 32 sequences from unknown archaea. The analysis of the 16S rDNA sequences of archaea collected from the bovine rumen, allows more sequences matching the unknown archaea were identified in the treatment with 70% hay.

A bactéria entomopatogênica Bacillus thuringiensis (Bt) é um agente de controle com características tóxicas e ambientais que permitem o controle de insetos-praga de acordo com as premissas do Manejo integrado de pragas (MIP). Com o objetivo de buscar novas linhagens potencialmente tóxicas para Sitophilus oryzae L. 1763 (Coleoptera: Curculinidae), caracterizaram-se molecularmente 1,073 isolados de B. thuringiensis de regiões do Brasil. O material genético foi extraído através do kit InstaGene Matrix, utilizado para a amplificação das seqüências através da técnica de Polymerase chain reaction PCR, sendo os resultados visualizados em gel de agarose 1,5%. A classe do gene cry35Ba foi representada por 60 isolados (5,6%) de Bt, os quais foram submetidos a bioensaio com larvas de S. oryzae. Quatro causaram mortalidade acima de 50% nos testes de patogenicidade e os isolados 544 e 622 foram os mais virulentos, conforme determinado pela estimativa da CL50. Nos quatro isolados que demonstraram toxicidade, foram detectados cristais esféricos, bipiramidais e cubóides, além de proteínas com 44 kDa, referentes aos genes cry35Ba por Sodium dodecil sulphate - polyacrilamide gel electrophoresis (SDS-PAGE). Estes dados demonstram o potencial de Bt no manejo de S. oryzae.

The entomopathogenic bacterium Bacillus thuringiensis is a control agent with toxic and environmental characteristics that allows the control of pest insects according to the Integrate Pest Management (IPM) precepts. In order to find new strains, potentially toxic to Sitophilus oryzae L. 1763 (Coleoptera: Curculinidae), 1.073 strains of B. thuringiensis from parts of Brazil were used. Genetic material was extracted with InstaGene Matrix kit, used for the amplification of sequences in Polymerase chain reaction (PCR), and viewed in 1.5% agarose gel. The gene cry35Ba class was represented by 60 B. thuringiensis isolates (5.6%), which were then subjected to bioassays with S. oryzae larvae. Among the isolates studied, four caused more than 50% mortality in pathogenicity tests, and the isolates 544 and 622 were the most virulent, as determined by CL50 estimates. The four toxic isolates had spherical, bi-pyramidal and cuboid crystals, and a 44-kDa protein was found in sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE), which coded for the product of cry35Ba genes. These data demonstrate the potential of B. thuringiensis for the management of S. oryzae larvae.

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.

Muitos genes estão envolvidos nos mecanismos de esporulação da bactéria Bacillus thuringiensis. A regulação e expressão desses genes resultam em uma produção massiva da proteína Cry, responsável pela morte das larvas de muitos insetos. Neste trabalho monitorou-se a expressão de genes de Bacillus thuringiensis, ao longo de três fases de seu desenvolvimento. Foram construídos macroarrays de DNA dos genes selecionados, cujas seqüências estão disponibilizadas no GenBank. Estes genes foram hibridizados com cDNAs obtidos de B. thuringiensis kurstaki HD-1. As sondas de cDNA foram sintetizadas a partir da transcrição reversa do RNA da bactéria, extraído durante as fases de crescimento logarítmico, estacionária e esporulativa, marcadas com 33PadCTP. A expressão diferencial encontrada foi significativa para dois genes de B.thuringiensis, um relacionado aos fatores sigma (sigma35) e outro ao gene cry (cry2Ab). Detectaram-se diferenças entre as médias de expressão do fator sigma e do gene cry2Ab. Os valores máximos de expressão diferencial foram obtidos para o gene codificador do fator sigma35 na fase log e na fase esporulativa. Na análise de médias observou-se expressão do gene cry2Ab apenas na fase log; no entanto, de forma bem mais baixa quando comparado com a expressão de sigma35, nas três fases.

There are several genes involved in Bacillus thuringiensis sporulation. The regulation and expression of these genes results in an upregulation in Cry protein production, and this is responsible for the death of insect larvae infected by Bacillus thuringiensis. Gene expression was monitored in Bacillus thuringiensis during three developmental phases. DNA macroarrays were constructed for selected genes whose sequences are available in the GenBank database. These genes were hybridized to cDNA sequences from B. thuringiensis var. kurstaki HD-1. cDNA probes were synthesized by reverse transcription from B. thuringiensis RNA templates extracted during the exponential (log) growth, stationary and sporulation phases, and labeled with 33PadCTP. Two genes were differentially expressed levels during the different developmental phases. One of these genes is related to sigma factor (sigma35), and the other is a cry gene (cry2Ab). There were differences between the differential levels of expression of various genes and among the expression detected for different combinations of the sigma factor and cry2Ab genes. The maximum difference in expression was observed for the gene encoding sigma35 factor in the log phase, which was also expressed at a high level during the sporulation phase. The cry2Ab gene was only expressed at a high level in the log phase, but at very low levels in the other phases when compared to the sigma35.