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

A
total of 2 x 106 parasites 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.