Clodronate Liposomes Depletion of Mosquito Hemocytes

The day prior to CSP_mut infection, naïve female mosquitoes (3–5 days old) were injected intrathoracically with either 69 nL of control liposomes or CLDs (Standard Macrophage Depletion Kit; Clodrosome^® + Encapsome^®; Encapsula Nano Sciences, Brentwood, TN, USA) using a Nanoject II injector (Drummond Scientific Co.). As previously described^{22 (link)}, 1:5 dilutions of liposomes and CLDs in 1 × PBS were used for the experiments. Following liposome injection, mosquitoes were infected with CSP_mut parasites. The effect of clodronate liposomes on the depletion of mosquito hemocytes was determined by the expression of cell markers NimB2 (hemocytes), LRIM15 (granulocytes), and SCRB9 (oenocytoids) with real-time PCR as previously reported^{26 (link)}. The change in oocyst melanization, the number of sporozoites, and relative expression of TEP1 were determined as described above.