Stingless bees are essential pollinators closely associated with microorganisms. Notably, Scaptotrigona sp. larvae acquire ergosterol—a hormone essential for pupation—by consuming Zygosaccharomyces sp. (Paludo et al., 2019). The mycelium grows within the brood cells and its consumption has been observed during in vitro rearing. Standardizing laboratory procedures involving this system is necessary for conservation studies.

Develop a simulated brood cell matrix for Scaptotrigona postica larval rearing and establish parameters for symbiotic fungus cultivation.

The matrix was developed using acrylic and microtitration plates with 92 and 300µL wells, respectively. The cerumen component was sterilized by autoclaving, melting at 60°C, or fragmented and transferred directly. Larval food samples were autoclaved by 120°C for 20 min., frozen at -80°C for 20 min. and thawed at 60°C for 3 min. or microfiltrated through a 0.45µm membrane; supplemented with 10% MgSO₄ or (NH₄)₂SO₄; and diluted in distilled water at 100%, 75%, or 50%.

During incubation, humidity was kept at 100% using distilled water, followed by 85% by using a KCl solution, and 75% with NaCl. Fungal biomass was quantified by centrifuging the samples at 2000g and weighing. Larval development (n=200) was assessed under the optimized conditions.

Highest fungal biomass values were obtained using the cerumen fragmentation approach and under the freeze-thawing condition (0.047g), without supplementation (0.039g) or with (NH₄)₂SO₄ (0.047g), and at 100% larval food concentration (0.035g). Zygosaccharomyces sp. growth was restricted to the regions of the wells coated with cerumen.

Larval rearing assays showed average pupation rates of 72% in microtitration plates and 8% in acrylic plates. The former proved to be more suitable for larval development when incorporating cerumen to the matrix, while the latter had higher mortality and malformation rates.

The study confirmed the viability of the simulated matrix for fungal growth and larval development, emphasizing the importance of replicating brood cell dimensions and components.

Paludo, C. R., Pishchany, G., Andrade-Dominguez, A., Silva-Junior, E. A., Menezes, C., Nascimento, F. S., Currie, C. R., Kolter, R., Clardy, J., & Pupo, M. T. (2019). Microbial community modulates growth of symbiotic fungus required for stingless bee metamorphosis. PLOS ONE, 14(7), e0219696. https://doi.org/10.1371/journal.pone.0219696