Plant or fungal sequences? An alternative optimized PCR protocol to avoid ITS (nrDNA) misamplification

The nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) from leaves of Drosera (Droseraceae) were amplified using "universal" primers. The analysis of the products demonstrated most samples were a molecular mixture as a result of unsuccessful and non-specific amplifications. Among the obtained sequences, two were from Basidiomycota fungi. Homologous sequences of Basidiomycota were obtained from GenBank database and added to a data set with sequences from Drosera leaves. Parsimony analysis demonstrated that one sequence was amplified from an Ustilaginomycetes fungus, and another from a Heterobasidiomycetes. Possibly these fungi were associated to leaves of Drosera, and not because of samples contamination. In order to provide optimization and a better specificity of PCR (polymerase chain reaction), a very successful method was demonstrated using dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) in reactions.

Os espaçadores internos transcritos do DNA nuclear ribossomal (ITS1 e ITS2) de folhas de Drosera (Droseraceae) foram amplificados com o emprego de iniciadores "universais". A análise demonstrou que a maior parte das amostras continha uma mistura resultante de amplificações não-específicas. Dentre as sequências de DNA obtidas, duas delas foram de fungos basidiomicetos. Sequências homólogas foram obtidas do GenBank e analisadas junto às sequências obtidas de folhas de Drosera. Através das análises filogenéticas de máxima parcimônia foi possível identificar uma seqüência como sendo de um Ustilaginomycetes e outra de Heterobasidiomycetes (Basidiomycota). Possivelmente esses organismos estavam associados às folhas de Drosera e assim não sejam resultantes de contaminação. Com o objetivo de otimizar e buscar uma melhor especificidade das reações de PCR, um protocolo bem sucedido foi demonstrado com o uso de dimetilsulfóxido (DMSO) e soroalbumina bovina (BSA).

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Identification and frequency of transposable elements in Eucalyptus

Transposable elements (TE) are major components of eukaryotic genomes and involved in cell regulation and organism evolution. We have analyzed 123,889 expressed sequence tags of the Eucalyptus Genome Project database and found 124 sequences representing 76 TE in 9 groups, of which copia, MuDR and FAR1 groups were the most abundant. The low amount of sequences of TE may reflect the high efficiency of repression of these elements, a process that is called TE silencing. Frequency of groups of TE in Eucalyptus libraries which were prepared with different tissues or physiologic conditions from seedlings or adult plants indicated that developing plants experience the expression of a much wider spectrum of TE groups than that seen in adult plants. These are preliminary results that identify the most relevant TE groups involved with Eucalyptus development, which is important for industrial wood production

Satellyptus: analysis and database of microsatellites from ESTs of Eucalyptus

The main goal of our research was to search for SSRs in the Eucalyptus EST FORESTs database (using a software for mining SSR-motifs). With this objective, we created a database for cataloging Eucalyptus EST-derived SSRs, and developed a bioinformatics tool, named Satellyptus, for finding and analyzing microsatellites in the Eucalyptus EST database. The search for microsatellites in the FORESTs database containing 71,115 Eucalyptus EST sequences (52.09 Mb) revealed 20,530 SSRs in 15,621 ESTs. The SSR abundance detected on the Eucalyptus ESTs database (29% or one microsatellite every four sequences) is considered very high for plants. Amongst the categories of SSR motifs, the dimeric (37%) and trimeric ones (33%) predominated. The AG/CT motif was the most frequent (35.15%) followed by the trimeric CCG/CGG (12.81%). From a random sample of 1,217 sequences, 343 microsatellites in 265 SSR-containing sequences were identified. Approximately 48% of these ESTs containing microsatellites were homologous to proteins with known biological function. Most of the microsatellites detected in Eucalyptus ESTs were positioned at either the 5 or 3 end. Our next priority involves the design of flanking primers for codominant SSR loci, which could lead to the development of a set of microsatellite-based markers suitable for marker-assisted Eucalyptus breeding programs

Isolation and maintenance of symbiotic fungi of ants in the tribe Attini (Hymenoptera: Formicidae)

The isolation and maintenance of symbiotic basidiomycete fungi living in association with ants of the tribe Attini has been hindered by the slow growth rate of these fungi and the presence of other microorganisms on the surface of the material which the ants maintain inside their nests to provide a growth substrate for their symbiont. In this paper we describe a method which increases the efficiency of isolation of these fungal symbionts by over seven fold as compared to traditional isolation procedures. Underground nests of attine ants of the genera Atta, Acromyrmex, Trachymyrmex and Mycetarotes were located, from which samples containing the fungal symbiont and ants were collected and transported to the laboratory where the ants were able to clean the fungal culture and stimulate its growth. As the symbiotic fungus grew, portions of its mycelium were collected and transferred to solid Yeast Nitrogen Base culture medium containing glucose and chloramphenicol. To facilitate the maintenance of the isolates in laboratory cultures, several nutrients were tested to formulate a complex culture medium for fast fungal growth and long-term storage. We successfully applied this methodology to the fungal symbionts of all the ant genera studied, thus producing a useful tool for the creation and maintenance of a comprehensive collection of fungi symbiotic of ants in the tribe Attini.

O isolamento e a manutenção de fungos basidiomicetos simbiontes de formigas da tribo Attini tem sido dificultado pela baixa velocidade de crescimento desses fungos, bem como pela presença de muitos microrganismos que vivem na superfície do material que as formigas mantêm no interior nos ninhos como substrato para o crescimento dos seus fungos simbiontes. No presente trabalho nós descrevemos um método que aumenta em mais de sete vezes a eficiência de isolamento desses fungos, quando comparada àquela obtida por procedimentos tradicionais. Ninhos subterrâneos de formigas atíneas dos gêneros Atta, Acromyrmex, Trachymyrmex e Mycetarotes foram localizados e deles foram coletadas amostras contendo fungos simbiontes e formigas, que foram transportadas para o laboratório, onde as formigas foram capazes de limpar a cultura do fungo e estimular o seu crescimento. Em seguida, porções dos micélios foram assepticamente coletadas e transferidas para meio Yeast Nitrogen Base contendo glicose e cloranfenicol. Para facilitar a manutenção dos isolados em culturas de laboratório, diferentes nutrientes foram analisados para a elaboração de um meio de cultivo complexo, que possibilitou aumentar a velocidade de crescimento dos fungos e estocá-los por longos períodos. O método foi aplicado com sucesso para os fungos simbiontes de todos os gêneros de formigas estudados, gerando, assim, um procedimento extremamente útil para a formação e manutenção de uma coleção representativa de diferentes fungos simbiontes de formigas da tribo Attini.

Occurrence of Winged Forms of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) in Laboratory Colonies / Ocorrência de Formas Aladas de Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) em Colônias de Laboratório

Foi observada, pela primeira vez no Brasil, a produção de centenas de formas aladas em formigueiros de Atta sexdens rubropilosa Forel, mantidos em laboratório. Larvas de último instar de machos foram encontradas em 5 de agosto de 2000 em um formigueiro com quase seis anos de fundação, contendo aproximadamente 110 L de esponja de fungo distribuída em 21 panelas. Entre 30 e 40 dias mais tarde apareceram larvas de fêmeas aladas. Os adultos sexuados apresentaram tamanhos compatíveis com os encontrados em formigueiros de campo. Foram observadas duas aparentes tentativas frustradas de revoada na última semana de outubro, após o que começaram a aparecer machos e fêmeas mortos no lixo do formigueiro.

The production of hundreds of reproductive forms of Atta sexdens rubropilosa Forel in nests maintained in laboratory was observed for the first time in Brazil. Last instar male larvae were found on August 5, 2000 in a six-year-old colony with approximately 110 L of fungus garden distributed in 21 pots. Thirty to forty days later, queen larvae started to be seen. The adult sexual forms were comparable in size with those found in the field. Two apparent failed attempts of a nuptial flight were observed during the last week of October, followed by the appearance of dead males and females in the garbage piles of the colony.