*2.1. Epimastigote Growth and Metacyclogenesis*

*Trypanosoma cruzi* (Dm28c strain) epimastigotes were grown in stationary phase at 28 ◦C in Brain Heart Infusion (BHI) medium supplemented with 10% FBS, 30 μM hemin, and 1% penicillin-streptomycin (P/S) cocktail (referred to hereafter as Regular Media, RM). Iron-Depleted Media (IDM) was prepared using an iron-free BHI medium, as described by Dick et al. [25]. Briefly, BHI medium without hemin addition was treated with Chelex (5 g/100 mL) for 1 h at room temperature and sterilized by filtration using 0.22 μm pore-size filters. To this Iron-Free BHI medium was added 1% P/S cocktail and 10% Iron-Free FBS. The iron-free FBS was prepared by adding 10 mM ascorbic acid for 6–7 h at 37 ◦C until the optical density at 405 nm had decreased by 50%. Then, the solution was supplied with 5 g of Chelex resin per 100 mL and incubated at room temperature under stirring at 50 rpm for 3–4 h, filtered to remove the resin, and dialyzed (with a cutoff of 2000 Da) against 4 l of cold, sterile PBS for 6 h, changing the solution every 2 h. The iron-free FBS was also sterilized by filtration using 0.22 μm pore-size filters and stored at −20 ◦C. Iron-Free FBS Iron-Depleted Media with Fe (IDM + Fe) was made the same way as IDM but with 8 μM Fe-citrate added. The parasites were inoculated (10<sup>6</sup> cells/mL) into the BHI medium on the sixth day of growth to test epimastigotes proliferation (RM, IDM, or IDM + Fe). Every day, cell proliferation was measured by counting the number of cells in a hemocytometer. Dm28c is a strain that differentiates "in vitro" and was previously used in our previous studies that demonstrated the existence of a Fe-reductase and a Fe-transporter in *T. cruzi* [25,29]. This strain is deposited in the Collection of Trypanosoma from Wild and Domestic Mammals and Vectors (COLTRYP), Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.

Metacyclogenesis was induced according to Koeller et al. [35]. Epimastigotes in the transition from the logarithmic to the stationary phase were adjusted to 5 × 108 parasites/mL in triatomine artificial urine (TAU) medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.035% (*w*/*v*) NaHCO3, and 8 mM phosphate buffer at pH 6.0). After 2 h at 28 ◦C, the cultures were diluted 100-fold in 10 mL TAU medium supplemented with 10 mM proline and 250 mM glucose (TAU-P) plus 500 g/mL G418 (Sigma-Aldrich, Saint Louis, MO, USA) and transferred to T25 flasks—lying at a 45◦ angle to increase the area in contact with O2—and kept at 28 ◦C to promote metacyclogenesis. Following 3–5 days, parasites were counted using hemocytometry, and the proportion of metacyclic epimastigotes (Tryp) was determined using their morphology after Giemsa staining.
