M7011

Ookinetes were counted by an adaptation of established protocols^{45 (link)}. At 20-26 h post bloodmeal, midguts were dissected from ten visibly blood-fed mosquitoes, placed in 20 μL 3% acetic acid, and vigorously pipetted to lyse midguts. An aliquot (2 μL) was loaded on a hydrophobic slide (VWR, 100488–892), spread to fill a 14 mm circle, dried, fixed with methanol, and stained with Giemsa. For each experimental group, the number of mature and immature ookinetes in sixty 1000X fields was recorded.
Oocyst and sporozoite counts were obtained by adaptation of established protocols⁴³ . At 9–10 d post feed, 15–30 midguts per group were dissected into 1X PBS on a hydrophobic slide (VWR, 100488-892). Samples were stained with 0.1% mercurochrome (M7011, Sigma Aldrich), covered, and examined (100X) for oocysts. Oocyst diameters were analyzed in ImageJ version 1.53. At 17–20 d post feed, salivary glands from 20-38 mosquitoes per group were collected, transferred to 100 μL 1X PBS on ice, sedimented (5000 g, 30 sec), and homogenized (sterile pestle, 30 sec). Parasites were counted by hemocytometer to obtain an average sporozoite count per mosquito.

Anopheles funestus females aged seven to ten days old (to allow for optimal mating and encourage blood feeding) were injected with 10 µg/µl of dsEcR or dsGFP as described previously. Ninety mosquitoes were included per replicate and a total of eight biological replicates were conducted. Subsequent to injection, the 10% sucrose solution was removed and replaced with distilled water to encourage blood feeding. Concurrently, mosquitoes were isolated for RNA extraction, DNase 1 treatment, cDNA synthesis and qPCR. Twenty-four hours after nanoinjection, mosquitoes were offered a PfNF54 infected blood meal (> 98% stage V gametocytes, 1.5−2.5% gametocytaemia, 50% (v/v) A + male human serum, Interstate blood bank Inc, Memphis, Tennessee, USA) for 40 min using a glass feeder. Unfed mosquitoes were discarded while fed mosquitoes were maintained on a 10% sucrose solution for eight days post feeding. After this time, mosquitoes were aspirated into ethanol to immobilize them and subsequently transferred to 1× PBS. Mosquito midguts were dissected 8 days post infection and stained on a microscope slide using 0.1% mercurochrome (M7011, Sigma, MO, USA) [44 ]. Midguts were subsequently viewed under a compound microscope between 20 and 40× magnification and the intensity and prevalence of oocysts in each midgut was counted and recorded.