‘Apparent’ sex ratio during gametocytogenesis.

<p>IFAT counts were performed on gametocyte preparations of stage II, stage III, stage IV, stage V, and 30 minutes after induction of activation (AG). Standard error is estimated for the ratio based on error calculated for the mean estimates of the proportion of males across the three independent cultures analysed.</p><p>% Alpha-tub. II+, % Pfg377+: indicate the percentage of gametocytes reacting with the given α-tubulin II and Pfg377 antibodies respectively.</p

Stage III and later gametocytes visualized with anti-Pfg377 and anti-α-tubulin II antibodies.

<p>Fluorescent staining of α-tubuIin (red) generated a characteristic striated pattern, particularly in earlier gametocytes. Expression of Pfg377 (green) was not seen prior to stage III. A) stage III; B) late stage III; C) stage IV; D) late stage IV; E) stage V; F) activated female gametocyte. Nuclear material was stained with DAPI, appearing blue in colour. Parasites were magnified ×1000.</p

Distinguishing between male and female gametocytes.

<p>A) IFAT image of stage V gametocytes showing all fluorophores from the secondary antibodies and DAPI (red, green and blue). B) Rhodamine (red) is visualized, which reacts with anti-α-tubulin II antibodies. C) DAPI (blue) is visualized, reacting with the nuclear material. D) FITC (green) is visualized, reacting with anti-Pfg377 antibodies. The white dotted circle shows gametocytes staining with anti-Pfg377 and anti-α-tubulin II (females), whereas the yellow dotted circle reveals one gametocyte that only reacts with anti-α-tubulin II antibodies (male). It can be seen that gametocytes which reacted with anti-Pfg377 antibodies, also reacted with anti-α-tubulin II antibodies, when comparing B) and D). Parasites were magnified ×1000.</p

Differential staining of gametocytes during gametocytogenesis.

<p>Pfg377 and α-tubulin II double-stained IFAT slides prepared on: A) day 2 (stage II); B) day 5 (stage III); C) day 7 (stage IV); D) day 11 (stage V); E) after gametocyte activation (AG). Parasites were magnified ×1000. Early stage parasites were more likely to be damaged during the purification process, and appear as stained with DAPI alone (panels A, B).</p

RT-PCR of sexual stage and sex specific proteins during gametocytogenesis.

<p>Transcripts of Pfs16, α-tubulin II and Pfg377 were amplified from preparations of stage I–V gametocytes. Gel electrophoresis of amplified products; + and − refer to presence or absence of reverse-transcriptase in the cDNA reaction prior to amplification. PC: positive control; NC: negative control. Cultures were not 100% synchronous. All samples (including positive controls) were run on a single gel at the same time. Lower bands (<100 bp) in each panel, particularly prominent in the absence of cDNA amplification, are primer dimers. Minor contamination of pfg377 DNA is visible in lanes 4, 8 and NC, lower panel.</p

Sex ratios established using light microscopy.

<p>Purified stage V gametocytes stained with Giemsa. A.) 3D7 magnet-purified gametocytes magnified ×1000. B.) (Detail) The cytoplasm of male gametocytes (MG) can be seen to stain pink and that of female gametocytes (FG) to stain purple; male gametocytes are smaller than females, the nucleus is bigger in males than in females, the granules of the malaria pigment are centrally located in female gametocytes and more widely scattered in males gametocytes The other 4 discriminatory characters can also be discerned.</p

MtDNA haplotypes and nucleotide diversity of <i>An</i>. <i>balabacensis</i> subpopulations based on <i>cox</i>1 sequences.

MtDNA haplotypes and nucleotide diversity of An. balabacensis subpopulations based on cox1 sequences.</p

Mitochondrial variation in subpopulations of <i>Anopheles balabacensis</i> Baisas in Sabah, Malaysia (Diptera: Culicidae) - Fig 3

Graphs of the mismatch distribution analysis for total populations of An. balabacensis based on (a) cox1, (b) cox2 and (c) the combined sequence. The dotted lines represent the observed frequency of pairwise differences, and the solid lines show the expected values for the population expansion model.</p

Mitochondrial variation in subpopulations of <i>Anopheles balabacensis</i> Baisas in Sabah, Malaysia (Diptera: Culicidae) - Fig 8

Variable positions of (a) nucleotide and (b) amino acid in the combined sequence of An. balabacensis. The numbers shown above the sequences represent the nucleotide or amino acid position and the dots refer to the identity with reference to ancestral haplotype (Hap_1).</p

Neutrality tests done on <i>An</i>. <i>balabacensi</i>s subpopulations.

Neutrality tests done on An. balabacensis subpopulations.</p