To normalize the number count of mitochondria and symbionts, a dilution curve was performed and the results obtained by Neubauer chamber counting were compared to the optical density (OD) on a wavelength of 600 nm. All the experiments were normalized to the medium efficiency by OD as 2.0 × 1010 for symbiont (OD = 0.9) and 4.5 × 108 for mithocondrion fraction (OD = 2.5). Protozoa were washed twice in PBS and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for 1 h. After being washed again in 0.1 M cacodylate buffer, pH 7.2 cells where postfixed for 1 h in 1% osmium tetroxide containing 0.8% potassium ferrocyanide, 5 mM CaCl2 in 0.1 M cacodylate buffer. Then, cells were washed, dehydrated in several crescent

concentrations of acetone and embedded in Epon: first a mix of Epon: Acetone (1 : 1) and finally pure Epon. Ultrathin sections were obtained in an Ultracut Reichert Ultramicrotome and mounted on 400 mesh copper grids, KU57788 stained with uranyl acetate and lead citrate. Samples were analyzed in a Zeiss 900 transmission electron microscope. Total lipids were extracted from A. deanei metabolically labeled after growth for 24,

36, and 48 h in the presence of [32Pi]-orthophosphate or from endosymbionts and mitochondria obtained after cell fractioning of protozoa treated see more or not with miltefosine for 24 h. Samples were washed with PBS, and the pellet was used for lipid extraction as described below. The lipid extraction was performed as described by Horwitz & Perlman (1987). Subsequently, the organic phase, containing the phospholipids, was solubilized with 3 mL of CHCl3 : CH3OH : HCl (200 : 100 : 0.75 v/v), and the phases were separated by centrifugation after addition of 0.3 mL of 0.6 N HCl. To purify the phospholipid over fraction, 0.5 mL of CHCl3 : CH3OH : HCl (3 : 48 : 47 v/v) was added to the organic phase and centrifuged. The pH was adjusted to 7.0 with 0.2 N NH4OH in methanol before dry under N2 gas. After lipid extraction, the protocol described by Einicker-Lamas et al. (1999) was used. Briefly, silica gel plates (Silica gel 60F254 Merck) were activated by heat, and the samples corresponding

to the lipid extracts of A. deanei, control and miltefosine-treated cells, grown in the presence of 32Pi, as well as lipid fractions derived from endosymbionts and mitochondria isolated from the host protozoan, were applied to the silica plates. The run of the samples was performed using a mobile phase (120 chloroform : 45 acetone : 39 methanol : 36 HCl : 24 H2O), as described by the method of Horwitz & Perlman (1987), for 80 min. The TLC plates were dried and exposed to develop in an iodine vapor atmosphere. Control standards (Sigma) were used to determine the phospholipids composition in each sample. When lipids were labeled by 32Pi, the TLC plate was sensibilized with 32Pi radiation, which was detected in Molecular Dynamics Storage Phosphor Screen GP after 24 h of exposure.