Journal: bioRxiv

Article Title: Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

doi: 10.1101/216721

Figure Lengend Snippet: To better understand factors critical for P. falciparum in vitro ookinete culture, we tested a wide range of conditions and assessed conversion rate. (A) Each experiment was compared with reference to our basic control protocol as conversion rates varied substantially between different gametocyte preparations. Each datapoint is an independent control culture (n = 79), line indicates the mean conversion rate and error bars denote the standard deviation.(B) P. falciparum NF54 strain was found to be significantly more efficient at fertilisation than 3D7 strain (P < 0.001, unpaired student’s T-Test). (C) The age of the culture had no impact on gametocyte fertility. (D) The impact of various factors on gametocyte fertility added to the developing gametocyte culture (pre-activation factors). Numbers on bars indicate the number of independent replicates performed and error bars denote the standard deviation. * indicates P < 0.05, unpaired student’s T-Test. (E) The impact of various factors on gametocyte fertility added to the parasites after ookinete culture induction (post-activation factors). Numbers on bars indicate the number of independent replicates performed and error bars denote the standard deviation. * indicates P < 0.05, unpaired student’s t-test.

Article Snippet: Parasites were incubated for 20 minutes with Cy3-conjugated mouse monoclonal antibodies 4B7 for P. falciparum (obtained through BEI Resources NIAID, NIH: Monoclonal Antibody 4B7 Anti- Plasmodium falciparum 25 kDa Gamete Surface Protein (Pfs 25), MRA-28, contributed by David C. Kaslow) or 13.1 ( ) for P. berghei (kindly provided by R. E. Sinden, Imperial College London), both at a concentration of 1:300 in ookinete medium containing Hoechst 33342 at a concentration of 1 μg/μl; thus identifying female gamete-ookinete specific proteins Pfs25 and Pbs28, respectively.

Techniques: In Vitro, Control, Standard Deviation, Activation Assay

Journal: bioRxiv

Article Title: Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

doi: 10.1101/216721

Figure Lengend Snippet: (A) By Giemsa staining, P. falciparum ookinetes isolated from the mosquito midgut show a slim, fluke-like shape, a compact nucleus and a darker apical end (left panels). In contrast, cells previously reported as cultured ookinetes (RCOs) had large elongated nuclei in the centre of the cell surrounded by pigment granules (right panel). Scale bar = 5µm. Representative images are shown from a minimum of triplicate experiment repeats. (B) By immunofluorescence, midgut ookinetes (Ook) express the female gamete/retort/ookinete marker Pfs25, whilst showing very faint staining for the male gametocyte/gamete marker 2606. In vitro , female gametes (Fem) and retorts (Ret) were also Pfs25 positive/2606 negative. In contrast, RCOs and exflagellation centres (Exfl) stained strongly for 2606, but not Pfs25. Scale bar = 12µm. Representative images are shown from a minimum of triplicate experimental repeats.

Article Snippet: Parasites were incubated for 20 minutes with Cy3-conjugated mouse monoclonal antibodies 4B7 for P. falciparum (obtained through BEI Resources NIAID, NIH: Monoclonal Antibody 4B7 Anti- Plasmodium falciparum 25 kDa Gamete Surface Protein (Pfs 25), MRA-28, contributed by David C. Kaslow) or 13.1 ( ) for P. berghei (kindly provided by R. E. Sinden, Imperial College London), both at a concentration of 1:300 in ookinete medium containing Hoechst 33342 at a concentration of 1 μg/μl; thus identifying female gamete-ookinete specific proteins Pfs25 and Pbs28, respectively.

Techniques: Staining, Isolation, Cell Culture, Immunofluorescence, Marker, In Vitro

Journal: bioRxiv

Article Title: Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

doi: 10.1101/216721

Figure Lengend Snippet: P. berghei (red bars) and P. falciparum (blue bars) gametocytes were induced to develop ookinetes in vitro (hatched bars). In parallel, the same parasite samples were also fed to mosquitoes to assess their intrinsic fertility in vivo (solid bars). At 26 hours post-activation, parasites were stained live with Cy3-conjugated antibodies specific for Pfs25 ( P. falciparum ) and Pbs28 ( P. berghei ). Conversion rates were determined as the percentage of retort/ookinete-shaped cells to round cells (unfertilised females). n = 6 paired experiments. Error bars denote the standard deviation. Significance determined by T-test.

Article Snippet: Parasites were incubated for 20 minutes with Cy3-conjugated mouse monoclonal antibodies 4B7 for P. falciparum (obtained through BEI Resources NIAID, NIH: Monoclonal Antibody 4B7 Anti- Plasmodium falciparum 25 kDa Gamete Surface Protein (Pfs 25), MRA-28, contributed by David C. Kaslow) or 13.1 ( ) for P. berghei (kindly provided by R. E. Sinden, Imperial College London), both at a concentration of 1:300 in ookinete medium containing Hoechst 33342 at a concentration of 1 μg/μl; thus identifying female gamete-ookinete specific proteins Pfs25 and Pbs28, respectively.

Techniques: In Vitro, In Vivo, Activation Assay, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

doi: 10.1101/216721

Figure Lengend Snippet: Representative flow cell sorting profiles of parasite cultures from a minimum of triplicate experimental repeats. Females were detected with 13.1-Cy3 anti-Pbs28 in P. berghei (A) and 4B7-Cy3 anti-Pfs25 in P. falciparum (B). Dot plots display cell subpopulations based on side-scattered light (SSC) and Cy3 intensity (561-562_15-A); the Cy3 surface positive female population is gated. Contour plots display subpopulations based on SSC and forward-scattered light (FSC), singlets (si) were selected within the female population specifically to exclude aggregated gametes, zygotes, retorts and ookinetes which adhere robustly to each other (31). Histograms represent the number of female parasites with a given Hoechst (405-450_50-A) profile. Light-blue represents Hoechst-unstained female parasites (background). Dark blue represents Hoechst stained female parasites. Fertilization events in wild-type P. berghei (WT) occur at high frequency but are rare in GEST knockouts (PbGEST-KO) (17), which display a high number of unfertilized events. Fertilization events in P. falciparum are low and most similar to the profile of P. berghei GESTKO.

Article Snippet: Parasites were incubated for 20 minutes with Cy3-conjugated mouse monoclonal antibodies 4B7 for P. falciparum (obtained through BEI Resources NIAID, NIH: Monoclonal Antibody 4B7 Anti- Plasmodium falciparum 25 kDa Gamete Surface Protein (Pfs 25), MRA-28, contributed by David C. Kaslow) or 13.1 ( ) for P. berghei (kindly provided by R. E. Sinden, Imperial College London), both at a concentration of 1:300 in ookinete medium containing Hoechst 33342 at a concentration of 1 μg/μl; thus identifying female gamete-ookinete specific proteins Pfs25 and Pbs28, respectively.

Techniques: FACS, Staining