2.11. Analysis of fungal virulence in Galleria mellonella

The G. mellonella larvae were from an in-house colony previously established (Clavijo-Giraldo et al., 2016) and were raised and fed ad libitum on corn bran and honey diet (1 kg corn bran, 150 g rice meal, 250 mL bee honey, and 70 mL glycerin) (García-Carnero et al., 2020). Only larvae with irritability, absence of body melanization, and a length of 1.2–1.5 cm were included in the host-fungus interaction assays (Clavijo-Giraldo et al., 2016, García-Carnero et al., 2020). Fungal inocula were injected in the last left pro-leg, previously sanitized with 70% (v/v) ethanol, with the assistance of a Hamilton syringe and a 26-gauge needle (Clavijo-Giraldo et al., 2016). The fungal inoculum was of 1 × 10⁵ yeast-like cells 10 µL ^-1. Larvae were kept in Petri dishes at 37 °C and survival monitored daily for 15 days. To maintain animal hydration, chopped apple was included in the animal housing during the observation period (García-Carnero et al., 2020). The animal silk was removed when evident to delay the transition to the pupa. Loss of irritability and extensive body melanization were taken as signs of insect death. Larvae were analyzed in groups that included 30 animals per experimental condition. As a control, one animal group was injected only with PBS, to assess animal mortality by animal manipulation or mechanical injuries To calculate the colony-forming units (CFUs), alive and dead animals were decapitated with a sterile scalpel, the hemolymph collected, serially diluted, and incubated on YPD plates, pH 4.5, at 28 °C for 72 h.

Hemocytes were quantified in anticoagulated hemolymph as previously reported (García-Carnero et al., 2020); whilst cytotoxicity and phenoloxidase activities were measured in cell-free hemolymph using 20 mM 3,4-dihydroxyDL-phenylalanine (Sigma-Aldrich) and the Pierce LDH Cytotoxicity Assay (Thermo Fisher Scientific), respectively (Martínez-Álvarez et al., 2019, Gómez-Gaviria et al., 2020).