Lack of correlation between micro fungi species and chemical control method of Atta treated with toxic baits

ABSTRACT: Atta 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.摘要：红切叶蚁（Atta sexdens rubropilosa）与真菌形成共生关系，同时由于其培育的真菌会被其他真菌寄生，因此与这类真菌存在负相互作用；此外，其菌圃中还存在若干功能尚未明确的其他真菌。本研究利用内转录间隔区（internal transcribed spacer，ITS）对经毒饵处理的蚁群中的真菌进行鉴定。本实验设置两种毒饵处理组：分别为含0.3%氟虫胺（sulfluramid）的0.75g毒饵，以及含0.003%氟虫腈（fipronil）的0.75g毒饵。采集真菌样本并接种于察氏培养基（Czapek medium）中培养7天，以促进真菌生长，便于后续鉴定。采用十六烷基三甲基溴化铵（CTAB）法从100~150 mg菌丝体中提取总DNA，利用通用引物ITS4与ITS5通过聚合酶链式反应（PCR）扩增ITS区。采用桑格测序法（Sanger method）完成测序。将所得序列进行BLAST比对，共鉴定出隶属于伞菌目（Agaricales）、散囊菌目（Eurotiales）、肉座菌目（Hypocreales）、格孢菌目（Pleosporales）、酵母菌目（Saccharomycetales）以及银耳目（Tremellales）的9个不同物种，序列同源性介于96%~100%。通过自动条形码间隔发现（Automatic Barcode Gap Discovery，ABGD）分析，共鉴定出9个类群，与NCBI数据库中已报道的物种一致。基于K2P距离采用邻接法构建系统发育树，结果显示各物种按照其系统发育类群进行聚类。本研究表明，切叶蚁蚁群中存在丰富多样的真菌，且本研究结果提示，分离得到的真菌物种与蚁巢所采用的防控方法之间不存在相关性。