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.

共收集2×10⁶个寄生虫，以1200×g离心10分钟使其沉淀，并用冰预冷的磷酸盐缓冲液（Phosphate Buffered Saline，PBS）洗涤一次，随后重悬于1ml血餐（绵羊血混合热灭活血清）中，采用鸡皮肤膜结合Hemotek喂食装置，于37℃条件下按前述方法饲喂昆虫。每组约80只雌性白蛉（Sand fly）取食1小时，随后选取60只完全饱血的个体，置于标准实验室条件下饲养至实验所需阶段。阴性对照组为仅饲喂绵羊血并按上述流程处理的白蛉。每日记录白蛉死亡率，于感染后第5天（Day Post Infection, DPI）解剖14只昆虫，此时昆虫已完成排便，寄生虫大概率已成功建立感染。寄生虫计数操作如下：将单个中肠解剖于PBS中，手动匀浆于50μl PBS内，随后采用血细胞计数板在显微镜下计数总寄生虫数。记录感染后第5天的白蛉感染率，并在实验全程每日评估白蛉存活率。本实验共设置3次生物学重复。