Assessing Fungal and Bacterial EV Toxicity in Galleria mellonella

The moth Galleria mellonella larvae were used to assess the potential toxicity of fungal and bacterial EVs according to the previously published protocols [29 (link)]. Larvae in their final instar stage (10 per group) were randomly chosen for the experiment and then inoculated in the last left proleg with 10 µl of the solution of fungal or bacterial EVs in sterile DPBS buffer, pH 7.50 ± 0.30 (Biowest) using a 10 μl Hamilton syringe (Merck). DPBS buffer served as a control for injection. The caterpillars were further incubated at 37 °C, and the number of dead larvae was scored daily; they were considered dead when they did not show movement in response to touch. The experiment was carried out in three biological replicates, and the averaged results are presented. The survival curves of EV-treated and control animals were compared using the Mantel-Cox log-rank test with GraphPad Prism 7.0. A value of p < 0.05 was considered significant.