D70 camera

Midgut smears of sand flies infected with Leishmania parasites were fixed with methanol, stained with Giemsa, examined under the light microscope Olympus BX51 and photographed with an Olympus D70 camera. Body length, flagellar length and body width of parasites were measured using Image-J software. Four morphological forms were distinguished as described in [21 (link)]: procyclic promastigotes (PP), elongated nectomonads (EN), metacyclic promastigotes (MP) and short promastigotes (SP). Haptomonads were not distinguished as these forms remain attached on the gut and their numbers are often underestimated on gut smears. In total, 200 promastigotes from four females/smears were measured for each Leishmania species.

Smears of sand fly guts infected by different Leishmania (S.) tarentolae strains were prepared on day 7 post blood meal. Morphometry of promastigotes on day 1 PBM was not performed as the numbers of parasites were too low. Gut smears were fixed with methanol, stained with Giemsa, and examined under the light microscope with an oil-immersion objective. Parasites were photographed with an Olympus D70 camera and measured using ImageJ software. Body length, body width, and flagellar length of 150 randomly selected promastigotes from at least three different sand flies were recorded (for each Sauroleishmania strain–sand fly combination). Criteria for morphological forms published by previous authors were modified [21 (link),38 (link),39 (link)] and following morphological stages were determined: (i) long nectomonads: body length ≥14 μm; (ii) short nectomonads: body length <14 μm and flagellar length <2 times body length; (iii) metacyclic promastigotes: body length <14 μm and flagellar length ≥2 times body length; (iv) rounded paramastigotes; and (v) haptomonads.

According to the above-described protocol, cells were incubated and treated for 24 h in 24-well plates with cover slides previously prepared following the protocol by Zjalic et al. [38 (link)] and coated with poly-D-Lysine (Sigma Aldrich, St. Louis, MO, USA). At the end of the experiment, cells were fixed with formaldehyde for 30 min at +4 °C. Afterwards, cells were washed 2x with PBS. Neutral lipids were stained using Oil-Red-O dye (ChemCruz, Huissen, The Netherlands). Fixed cells were stained with a working Oil-Red-O solution for 30 min. Oil-Red-O 0.5% stock solution was dissolved in isopropanol, and the working solution was prepared as 60% Oil-Red-O stock solution with 40% water. Fixed cells were stained with a working Oil-Red-O solution for 30 min. After rinsing two times with phosphate buffered saline (PBS), cells were mounted in the fluorescent mounting medium with 4′,6′-diamidino-2-phenylindole (DAPI) (Abcam, Cambridge, UK). The cells were visualized using a microscope (Axioskop 2 MOT Inverted microscope, Carl Zeiss, Göttingen Germany) with an Olympus D70 camera, controlled through the computer program DP Manager 1.2.1.107 and DP Controller 1.2.1.108. ImageJ-Fiji software was used to count cell nuclei and measure integrated density relative to the cell count. Results are shown as a percentage relative to the negative control of at least three biological replicates.