LAUSR

EnglishAtta sexdens rubropilosa (leaf-cutter ants) has a symbiotic association with a fungus and has a negative interaction with other fungi due to parasitism of the fungus cultivated by ants; also, there are several other fungi with no exact known role occurring in their cultivated fungus garden. In the present study, we use the ITS region (internal transcribed spacer) to identify fungi in colonies treated with toxic baits. Experiments using two toxic baits were carried out: 0.75g of sulfluramid [0.3%] and 0.75g fipronil [0.003%]. Samples of fungi were collected and cultured in Czapek medium for seven days to allow fungal growth and subsequent identification. Total DNA was isolated from 100-150 mg of mycelium using the CTAB method and using PCR, with the universal primers (ITS4 and ITS5), to amplify the ITS region. Sequencing was performed using the Sanger method. Sequences were subjected to BLAST, allowing the identification of nine different species of the orders Agaricales, Eurotiales, Hypocreales, Pleosporales, Saccharomycetales and Tremellales showing a variation in identity of 96-100%. Using “The Automatic Barcode Gap Discovery” analysis, nine groups were identified, corresponding to species described in NCBI. The K2P distances were used to generate a tree using Neighbour-joining, demonstrating that the species were grouped according to phylogenetic groups. We concluded that leaf-cutter ant colonies exhibited a wide variety of fungi and this study suggested that there is no correlation between the species of fungi isolated with the control method used on the ant nest. Key words: ITS; DNA barcoding; leaf-cutter; ants portuguesAtta sexdens rubropilosa (cortadeira de folha) possui associacao simbiotica com fungos e interacao negativa com outros fungos devido ao parasitismo do fungo cultivado pelas formigas. Quando colonias da formiga cortadeira de folhas sao submetidas ao tratamento com iscas toxicas, diversas especies de fungos surgem dentro da colonia, podendo contribuir com a morte ou sobrevivencia da colonia. Para entender os relacionamentos ecologicos em colonias de formigas, a identificacao de especie de fungos se torna muito importante e, o uso de DNA barcoding tem sido um metodo rapido e eficiente para identificacao de especies usando metodos moleculares. No presente trabalho, usamos a regiao ITS (internal transcribed spacer) para identificar fungos em colonias tratadas com iscas toxicas. Dois experimentos com iscas toxicas foram aplicados: 0.75g de Fipronil [0.003%] e 0.75g de Sulfluramid [0.3%]. As amostras, contendo os possiveis fungos, foram coletadas e cultivadas em meio Czaped durante sete dias para o crescimento do fungo e posterior identificacao. O DNA total foi isolado de 100-150mg de micelio usando o metodo CTAB, usado para amplificar a regiao ITS por PCR empregando primers universais (ITS5 e ITS4). O sequenciamento foi realizado utilizando o metodo de Sanger. As sequencias foram submetidas ao BLAST, permitindo identificar nove diferentes especies das ordens Agaricales, Eurotiales, Hypocreales, Pleosporales, Saccharomycetales e Tremellales, mostrando variacao 96-100% de identidade. Empregando a analise “The Automatic Barcode Gap Discovery”, identificou-se nove grupos, correspondendo as especies descritas no NCBI. As distâncias K2P foram usadas para gerar uma arvore usando Neighbour-Joining, apresentando que as especies foram agrupadas de acordo com as filogenias dos grupos. Conclui-se que as colonias de formigas cortadeira de folhas apresentam grande diversidade de fungos e que DNA barcoding e eficiente para identificacao destes. Palavras-chave: ITS; DNA barcoding; formigas cortadeira; Atta; ecologia