A see more total of 10,000 events were acquired in the region previously established as that corresponding to the parasites. All analyses were performed in at least 3 independent experiments. The T. cruzi epimastigotes and trypomastigotes were treated with the melittin peptide (1.22–4.88 and 0.07–0.28 μg/ml, respectively) or not (control cells) for 24 h, washed with PBS (pH 7.2) and incubated in the dark with 100 μM of monodansyl cadaverine (MDC) (Sigma–Aldrich) for 1 h at 28 °C (epimastigotes) or 37 °C (trypomastigotes). The parasites were then washed twice in PBS and fixed with freshly prepared 2% formaldehyde for 20 min
at room temperature. Each condition was performed in triplicate (100 μl final volume) in a black 96-well plate and analyzed in a Molecular Devices Microplate check details Reader (a SpectraMax M2/M2e spectrofluorometer) using 355 and 460 nm wavelengths for excitation and emission, respectively. The suspensions of 2% FA and 2% FA plus 100 μM
MDC alone were used as reaction controls and were simultaneously read in the plate. The mean value comparisons between the control and treated groups were performed using the Kruskal–Wallis test with the BioEstat 2.0 program for Windows. The differences with p values ≤0.05 were considered statistically significant. The epimastigotes were grown for 4 days in LIT medium containing different concentrations of melittin, and the percentage of surviving parasites was evaluated (Table 1). The IC50 (50% growth inhibition) after 24 h of treatment was 2.44 ± 0.23 μg/ml. Because the trypomastigote forms do not multiply, the cytolytic effect of the venom on trypomastigotes was evaluated after 24 h of treatment. The LD50 of melittin for the trypomastigotes
was 0.14 ± 0.05 μg/ml Loperamide (Table 1). The morphological alterations of the epimastigotes (Fig. 1) and trypomastigotes (Fig. 2) induced by 1 day of treatment with 2.44 and 0.14 μg/ml of melittin, respectively, were observed by SEM. Most of the treated parasites presented with swollen and abnormal cell body conformations (Fig. 1 and Fig. 2B, C) as compared to the control cells (Figs. 1A and 2A). Occasionally, a complete alteration of the parasite shape was observed (Figs. 1B and 2B, C). Some epimastigotes also presented with altered flagellar morphologies, which appeared to be cracked, lumpy and occasionally broken in appearance (Fig. 1B, C). The trypomastigotes presented with plasma membrane blebbing and membrane disruption with cytoplasmic overflow, indicating severe membrane disorganization (Fig. 2B, C). The ultrastructural alterations caused by melittin were also analyzed using TEM (Figs. 1 and 2). The treated epimastigotes showed an intense swelling of the mitochondria (Fig. 1E, F), with an altered inner mitochondrial membrane that formed concentric membrane structures within the organelle (Fig. 1F).