Fig. 2. Characteristics of the phenotype of Dko and wt ookinetes during ookinete maturation, penetration of the midgut epithelium and development into oocysts. (A–D) Light microscopic morphology of in vitro cultured ookinetes in Giemsa-stained smears. (A) wt ookinetes form characteristic clusters, which are absent in Sko and Dko ookinete cultures (Table I). (B and D) Dko parasites (D) have in general a ‘swollen’ appearance (both in smears and in live preparations) compared with wt (B) and Sko ookinetes. However, no changes in membrane integrity could be demonstrated by electron microscopy in these ookinetes as a result of the lack of P25 and P28 (results not shown). (C) P25ko ookinetes attached in a typical ‘floret’ to mononuclear cells in culture after trypsin treatment. Scale bar = 10 µm. (E) RT–PCR analysis of the expression of the immune response genes GNPB and defensin in A.gambiae in refractory (r) and susceptible (s) lines 24 h after feeding on uninfected (N) or infected (I) mice (see Materials and methods). The immune responses against infection with 25/28Dko and wt are compared. The clear immune response in both the refractory and susceptible strain after infection with Dko parasites demonstrates that in both strains ookinetes are able to penetrate the midgut wall. RT–PCR analyses of mRNA of genes encoding mosquito ribosomal protein S7, P25, P28 and CTRP are used as controls. (F and G) Dko ookinetes within the midgut epithelium stained with antibodies recognizing CTRP in the susceptible (F) and refractory (G) strain of A.gambiae. Scale bar = 10 µm. (H and I) Melanized, penetrated ookinetes on the surface (haemocoel side) of the midgut epithelium of the refractory strain of A.gambiae. (H) 25/28Dko; (I) wt. Scale bar = 50 µm. (J–L) Melanized wt (J) and Dko (K) ookinetes emerging from midgut epithelia of the refractory strain of A.gambiae and a non-melanized Dko ookinete, which are typically found deep within the midgut epithelia of refractory A.gambiae. Scale bar = 10 µm. (M and N) wt (M) and Dko (N) oocysts (10 days old) isolated from midguts of A.stephensi that are infected by micro-injection of ookinetes into the haemocoel. In wt infected mosquitoes, oocysts are often found in clumps attached to the midgut basal lamina, whereas these clumps are lacking in Dko parasites and oocysts are often found free in the haemocoel. Scale bar = 50 µm.