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. 2007 Aug;116(4):504–508. doi: 10.1016/j.exppara.2007.01.015

Fig. 3.

Fig. 3

Plasmodium berghei Δpplp5 ookinetes fail to invade and cross the Anopheles stephensi midgut. (a–c) A. stephensi midguts were dissected 24 h after feeding of wt or Δpplp5 parasites and epithelia were prepared as previously described (Danielli et al., 2000). Sheets were incubated with purified rabbit α-AgSRPN6 (1:1000) and monoclonal α-Pbs28 (13.1; 1:1000) antibody followed by secondary Alexa-Fluor-488-labelled-goat anti-mouse IgG and secondary Alexa-Fluor-568-labelled-goat anti-rabbit IgG (1:1500, Molecular Probes). Cell nuclei were stained with TO-PRO-3 (Molecular Probes). Samples were analysed using a Leica SP2 confocal (b,c) or a Leica DMR fluorescence microscope and Leica DC500 digital camera (a). Scalebar = 20 μm. (a) Differential interference contrast images show extruding midgut cells (white arrowhead) following invasion by wt ookinetes (left), and undamaged gut with large numbers of attached Δpplp5 ookinetes (black arrowhead) (right). (b) Confocal 3D projection of a z-stack shows that wt ookinetes have successfully invaded the midgut and started rounding up (green; white arrowhead; left) resulting in upregulation of A. stephensi Serpin 6 (red). No Serpin expression was detected in mosquitoes fed with Δpplp5 parasites (white arrowhead; right). (c) z-Stacks show that wt ookinetes (green) have crossed the midgut epithelium (top) while Δpplp5 ookinetes (green) are still found on the apical side (bottom). Open arrowheads indicate nuclei of hemocytes, which are found attached to the basal side of the midgut epithelium. (d) A. stephensi midguts were dissected 24 h after feeding of wt or Δpplp5 parasites, fixed as described in Sinden et al. (1985) and semithin sections (500 nm) were prepared and stained with toluidine blue. Images were taken using a Leica DMR fluorescence microscope and Zeiss AxioCam digital camera. Scalebar = 50 μm. Invasion by wt ookinetes has caused massive damage to the midgut epithelium (ME) (left), while midguts of mosquitoes fed with Δpplp5 remain unharmed (right, two examples shown). wt ookinetes (arrowhead) are found within the midgut epithelium (left), whereas Δpplp5 ookinetes (arrowhead) have successfully crossed the peritrophic matrix (PM) but are stuck within the microvilli layer (MV) (right, two examples shown). BM, blood meal.