Non-invasive visualisation and identification of fluorescent Leishmania tarentolae in infected sand flies

A total of 2 x 10⁶parasites were pelleted (1200xg, 10 min) and washed once with ice-cold PBS before resuspending in 1ml of blood meal (sheep blood mixed with heat-inactivated serum) and fed to the insects using a chick skin membrane with the Hemotek apparatus at 37 °C as described above. Groups of ~80 females were fed for one hour, after which 60 fully-engorged individuals were separated and kept under standard laboratory conditions until required for experimental work. Negative control groups were flies fed only on sheep blood and manipulated as stated above. Sand fly mortality was recorded daily and 14 insects were dissected 5 days post infection (DPI), when defecation had already taken place and parasites have likely established an infection. Parasite counts were performed by dissecting individual midguts in PBS and manually homogenizing them in 50 ul of PBS. Total parasites were counted under the microscope using a haemocytometer. Percentage of infected flies 5 DPI was recorded and sand fly survival was evaluated every day for the duration of the experiment. Three biological replicates were performed.