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Phylogenetic, morphological and chemical analyzes of Zygosaccharomyces associated with stingless bees

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Author(s):
Gabriela Toninato de Paula
Total Authors: 1
Document type: Master's Dissertation
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
Defense date:
Examining board members:
Monica Tallarico Pupo; Cristiano Menezes; Carlos Augusto Rosa
Advisor: Monica Tallarico Pupo
Abstract

Social insects are known to establish symbiosis with microorganisms. Their colonies provide microenvironments suitable for microbial growth, such as stable environmental conditions and rich source of food. In return, insects can benefit from nutrients and/or protection against pathogens. However, little is known about this type of mutualistic interaction involving bees. Only recently a symbiotic interaction has been described between the stingless bee Scaptotrigona depilis and the fungus Zygosaccharomyces sp. This fungus grows inside the brood cells of S. depilis and is ingested by the larvae, being indispensable for their survival and development. This is due to the fungus\' ability to accumulate lipids in adiposomes, including the steroid ergosterol, which is used by S. depilis larvae during their metamorphic process. In the present study, the objective was to verify whether the same phenomenon also occurs in other species of stingless bees. The objective was to verify if there is the presence of symbiotic food fungus and if it is a source of steroids for larval development. For this, samples were obtained from the brood cells of 19 species of stingless bees belonging to 12 different genera. Zygosaccharomyces spp. was isolated from eight species. In addition to the brood cells, it was also possible to obtain Zygosaccharomyces spp. from other parts of the colony, such as honey, pollen, adult insect and refuse/garbage, which made it possible to make comparative analyzes. Image analysis by optical microscopy demonstrated that Zygosaccharomyces spp. strains isolated from brood cells are capable of forming pseudohyphae, while the others isolated from other sites in the colony presented spherical and/or ovoid cells. Genetic sequencing of the D1/D2 regions of the 26S and 18S rRNA gene allowed the construction of three phylogenetic trees. The phylogenetic analyses showed the formation of a clade made up only of Zygosaccharomyces spp. strains isolated from the brood cells of different stingless bee species. These results highlight the specificity of the fungus for this system involving stingless bees. The analysis of steroids and ergosterol quantification by GC-MS was carried out for the strains identified as Zygosaccharomyces spp., isolated from the brood cell and other colony sites, as well as from other filamentous fungi isolated sporadically from the brood cells of some bees. The GC-MS data demonstrated that there is no different pattern in the concentration of this steroid among the strains. Altogether, the results indicate that the association of Zygosaccharomyces seems to be a frequent symbiotic interaction in stingless bees. The determination of the association of these microorganisms in this system provides subsidies for additional studies regarding the use of fungicides in agriculture since they can indirectly affect the development of these bees. (AU)

FAPESP's process: 18/03650-0 - Analysis of steroids produced by symbiotic fungi isolated from the stingless bees' rearing cell
Grantee:Gabriela Toninato de Paula
Support Opportunities: Scholarships in Brazil - Master