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.

将总计2×10⁶个寄生虫以1200×g离心10分钟进行沉淀，采用冰态磷酸盐缓冲液（PBS）洗涤一次后，重悬于1ml血餐（绵羊血混合热灭活血清）中，使用Hemotek饲喂装置（Hemotek apparatus）辅以鸡皮膜，于37℃条件下喂养白蛉，操作流程如前文所述。每组约80只雌性白蛉取食1小时，随后分离出60只完全吸血的个体，置于标准实验室条件下饲养，直至用于后续实验。阴性对照组为仅饲喂绵羊血并按前述方法处理的白蛉。每日记录白蛉的死亡率；于感染后5天（DPI，post infection）解剖14只白蛉，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：将单只白蛉中肠解剖于PBS中，手动将其匀浆于50μl PBS内，随后在显微镜下使用血细胞计数板（haemocytometer）统计总寄生虫数量。记录感染后5天时的白蛉感染率，并在实验全程每日评估白蛉存活率。本实验共完成3次生物学重复（biological replicates）。