Yeasts from the nests of two Amazonian stingless bees: screening and PCR-RFLP molecular analysis

The Amazonian Forest hosts a great diversity of stingless bees. The Tropical Amazonian climate – moist and hot – favors the development of a diverse mycobiota associated with them, which is largely unknown. We isolated and identified yeasts associated with the nests of Melipona interrupta and Cephalotrigona femorata , which are native to the Central Amazonia. The yeasts were cultured on YPD and screened with a BD ™ CHROM-agar ™ Candida medium (CHROM-agar). Isolates with the same color pattern were identified based on the sequencing of the hypervariable domains from rRNA loci (ITS1/2 and 28 S rRNA D1/D2) and the Dde I restriction pattern targeting 3Kb amplicons from the same loci via the PCR-RFLP method. We isolated 532 yeasts from the nests of pollen, honey, larval food, and cerumen, which were grouped into 15 color and restriction pattern. The 15 strains belonged to the genera: Aureobasidium , Candida , Debaryomyces , Hyphopichia , Hanseniaspora , Kodamaea , Metschnikowia , Pichia , Rhodotorula , Trichosporon , Wickerhamiella , and Zygosaccharomyces . The combined use of the YPD cultivation method and CHROM-agar medium screening allowed the identification of the diverse community of yeasts associated with these stingless bees, while Dde I restriction pattern analysis enabled the discrimination of the strains in 15 different yeast groups and can serve as a model for further studies.