Bacteria, yeasts, and fungi associated with larval food of Brazilian native stingless bees

Abstract

Stingless bees are a diverse group with a relevant role in pollinating native species. Its diet is rich in carbohydrates and proteins, by collecting pollen and nectar supplies the development of its offspring. Fermentation of these products is associated with microorganisms in the colony. However, the composition of microorganisms that comprise this microbiome and its fundamental role in colony development is still unclear. To characterize the colonizing microorganisms of larval food in the brood cells of stingless bees Frieseomelitta varia, Melipona quadrifasciata, Melipona scutellaris, and Tetragonisca angustula, we have utilized molecular and culture-based techniques. Bacteria of the phyla Firmicutes, Proteobacteria, Actinobacteria, and fungi of the phyla Ascomycota, Basidiomycota, Mucoromycota, and Mortierellomycota were found. Diversity analysis showed that F. varia had a greater diversity of bacteria in its microbiota, and T. angustula had a greater diversity of fungi. The isolation technique allowed the identification of 189 bacteria and 75 fungi. In summary, this research showed bacteria and fungi associated with the species F. varia, M. quadrifasciata, M. scutellaris, and T. angustula, which may play an essential role in the survival of these organisms. Besides that, a biobank with bacteria and fungus isolates from LF of Brazilian stingless bees was created, which can be used for different studies and the prospection of biotechnology compounds.

Figure 1

(A) Venn Diagram of ASVs classified for stingless bees; (B) Genus × Family histogram of 15 genera most abundant.

Figure 2

(A) Venn Diagram of ASVs classified for stingless bees; (B) Genus × Family histogram of 20 genera most abundant; (C) Species × Genera histogram of 22 species most abundant.

Figure 3

Alpha diversity index generated from the data of Bacterial (A) and Fungal (B) community sequencing.

Figure 4

PCoA analysis Bacterial (A) and Fungal (B) community sequencing.

Figure 5

(A) Abundance of genera bacterial isolated from larval food of stingless bees. (B) An abundance of species was found from the larval food of stingless bees.

Figure 6

Methodology for cultivating larval food from four stingless bee species with different dilutions and culture mediums. Brain Heart Infusion (BHI), Nutrients (NUT), Tryptone soy agar (TSA), Man, Rogosa and Sharpe (MRS), Mac Conkey (MCK), Mannitol Salgado (MAS), Yeast Malt Agar (ISP), Potato Dextrose Agar 2% (PDA2) and 20% (PDA20) glucose, PDA acidified with tartaric acid (PDAa), PDA NaCl 5% (PDA5s), Oatmeal Agar (OAT), Yeast Extract-Peptone-Dextrose (YPD), DRBC (Dichloran Rose-Bengal Chloramphenicol) e Sabouraud (SAB). Images of representative method from ‘Smart Servier Medical Art’ (https://smart.servier.com/).