Figure 1.
Oocyst and Sporozoite Development of wt and Δpblap Parasites
Graphical summary of oocyst numbers (A), midgut sporozoite numbers (B), and salivary gland sporozoite numbers (C) of wt and Δpblap parasites. Values of Δpblap parasites are given as mean % of wt (± standard error of the mean). Δlap1 as published in [2]. Please refer to Tables S3 and S4 for individual data.
Figure 2.
Oocyst Morphology of wt and Δpblap Parasites
Differential interference contrast images of wt day 21 p.i. (A), Δpblap2 day 13 p.i. (B), Δpblap4 day 18 p.i. (C), and Δpblap6 day 21 p.i. oocysts (D) in An. stephensi. Most wt oocysts have undergone sporulation (open arrow). No sporozoite formation is observed in Δpblap infections, and oocysts appear either immature/enlarged (open arrowhead) or degenerate/vacuolated (closed arrowhead). Some Δpblap4 oocysts are melanized (closed arrow). Scale bar = 20 μm.
Figure 3.
Transmission Electron Micrographs of wt and Δpblap Oocysts
All images taken on day 13 p.i. unless otherwise indicated. Scale bar = 1 μm (A, B, F) or 5 μm (C–E). ep, midgut epithelium.
(A) wt oocyst showing normal morphology of the endoplasmic reticulum (er).
(B) Δpblap2 oocyst showing extensive expansion of the endoplasmic reticulum (er).
(C) Δpblap1 oocyst (day 27 p.i.) showing extensive expansion of the endoplasmic reticulum (er) and some budding sporozoites (s).
(D) wt oocyst showing normal morphology following cytokinesis to produce hundreds of daughter sporozoites (s).
(E) Δpblap2 oocyst showing extensive degeneration and few nuclei (some of which are labelled n).
(F) Degenerate Δpblap4 oocyst showing prominent melanization (m) of the extracellular oocyst wall (cw) which appears to spread into the mosquito basal lamina (bl).
Figure 4.
Sporozoite Development in Genetic Crosses
Graphical summary of salivary gland sporozoite numbers derived from crosses between wt and Δpblap and amongst Δpblap strains. Values given are mean % of wt (± standard error of the mean). In wt crosses, diagnostic PCR on blood stage infection resulting from mosquito bite confirmed transmission of the Δpblap parasites (not shown). Δlap1 as published in [4]. Please refer to Table S5 for individual data.
Table 1.
Genetic Crosses between Δpblap and Δpbs47 or Δpbs48/45
Figure 5.
RT-PCR Analysis of pblap Expression in Day 10 Oocysts
RT-PCR on total RNA isolated from midguts of An. stephensi infected with Δpblap1 × Δpbs47 (lanes 1 and 2), Δpblap2 × Δpbs47 (lanes 3 and 4), Δpblap4 × Δpbs47 (lanes 5 and 6), Δpblap6 × Δpbs47 (lanes 7 and 8), Δpblap1 × Δpbs48/45 (lanes 9 and 10), Δpblap2 × Δpbs48/45 (lanes 11 and 12), Δpblap4 × Δpbs48/45 (lanes 13 and 14), and Δpblap6 × Δpbs48/45 (lane 15 and 16). Top panel, PCR for the respective pblap gene as indicated above the panel; bottom panel, control PCR for a-tubulin; RT, reverse transcriptase. Fragments of the expected size were amplified for pblap2 (399 bp), pblap4 (482 bp), pblap6 (381 bp), and α-tubulin (432 bp). A weak signal for pblap1 (540 bp) was observed on longer exposures in both crosses. Some genomic DNA contamination was detected for Δpblap6 × Δpbs47 (lane 8, bottom panel), but the pblap6 PCR product from cDNA (lane 7, top panel) can be distinguished from amplification of genomic DNA by the absence of a 310-bp intron.