Dm6000 wide field fluorescence microscope

Bright field images were taken and quantified using Lucia imaging software and a Leica FW4000 upright microscope equipped with a SPOT digital camera. Fluorescence images were obtained using a Leica DM6000 wide field fluorescence microscope equipped with a Leica FX350 camera with 20× and 40× objectives. Images were taken through several z-sections and de-convolved using Leica software. A Leica TCS SP2 confocal laser-scanning microscope was used with 40× and 63× objectives to acquire high-resolution images.

The interactions between An. gambiae and Plasmodium parasites were assayed using the rodent malaria model, P. berghei. The following reagent was obtained through BEI Resources, NIAID, NIH: Plasmodium berghei, Strain (ANKA) 507m6cl1, MRA-867, contributed by Chris J. Janse and Andrew P. Waters. Low passage P. berghei frozen stocks were serially passaged in 6–8 week-old Swiss Webster mice by intraperitoneal injection. Mouse blood parasitemia and microgametocyte exflagellation were assessed by microscopy prior to mosquito blood feeding. Mosquitoes were fed on anesthetized mice carrying a 2–6% parasitemia 3 d post-infection. Mosquitoes were fed at 21°C and held at this temperature for the duration of the experiment. Midguts were isolated from mosquitoes 9–10 d post-bloodmeal, incubated in 4% paraformaldehyde for 60–90 min at room temperature, rinsed in 1X PBS, and mounted in Vectashield (Vector Laboratories). GFP-positive oocysts and melanized ookinetes were manually counted and imaged with a Leica DM6000 widefield fluorescence microscope. Raw oocyst and ookinete counts are provided in the Supporting Information (S1 Dataset).