DHHC10 is essential for transmission to the mosquito vector. (A) RNA sequencing data from blood and mosquito stages for all 11 P. berghei dhhc genes reveal that dhhc2, dhhc3, and dhhc10 are up-regulated in gametocytes (). Error bars correspond to SEM values. FPKM, fragments per kilobase of transcript per million mapped reads. (B) RT-PCR of dhhc3 and dhhc10 confirm the gametocyte-specific nature of dhhc10, whereas dhhc3 is also transcribed in asexual stage parasites. RT+, RT-positive reaction; RT−, RT-negative reaction. (C) Schematic representation of rodent and human malaria DHHC10 with four TM domains. The signature DHHC motif is located between TM domains 2 and 3. Drawn to scale. (D) Δdhhc10-a parasites develop WT oocyst numbers at day 12–13 postinfection. Absolute numbers of oocysts/midgut from five independent experiments are presented for both WT (n = 70) and Δdhhc10-a (n = 48) parasites. Mean ± SEM values are shown; P values are for the Mann–Whitney test. (E) Δdhhc10-a oocysts appear normal-sized at day 14 postinfection but lack signs of sporulation and remain empty (arrowheads), whereas WT oocysts have already formed sporozoites (arrows). (Scale bar: 20 µm.) (F) Immunofluorescence of oocyst-infected midguts at day 14 postinfection reveals strongly reduced expression of the developmental marker CSP in Δdhhc10-a parasites; DNA staining (Hoechst 33342) is comparable. ↑CSP indicates long exposure. (Scale bar: 20 µm.) (G) Western blot analyses of oocyst-infected midguts at day 13 postinfection confirms low CSP expression in Δdhhc10-a mutants. Here HSP70 served as a loading control. ↑CSP indicates overexposure. (H) Δdhhc10-a parasites do not colonize the mosquito salivary glands. WT, five independent experiments, n = 15; Δdhhc10-a, four independent experiments, n = 10. Mean ± SEM values are shown. (I) Mice bitten by Δdhhc10-a–infected mosquitoes fail to develop blood stage infections. Mean ± SEM parasitemias from three independent experiments are shown.

dhhc10 is translationally repressed in gametocytes and translated in ookinetes, where it localizes to the crystalloid. (A) Only dhhc10::gfp mRNA is transcribed in dhhc10::gfp parasites. p28 served as the control gene for RT-PCR. (B) DHHC10::GFP is translated only in ookinetes. DNA was stained with Hoechst 33342. (Scale bar: 5 µm.) (C) RT-PCR analyses of DOZI::GFP and CITH::GFP gametocyte IP eluates showing that dhhc10 mRNA, like p25 and p28 mRNAs, is bound by both translational repressors. Translated mRNAs (dozi, cith, and alba-3) are not enriched in the IP-GFP fractions. Input, total gametocyte mRNA; IP-GFP, IP with anti-GFP antibody; IP-c-myc, IP with anti–c-myc antibody; IP beads, no antibody used for IP. (D) DHHC10::GFP colocalizes with the crystalloid-resident protein LAP3 (here tagged with mCherry). (Inset) A higher-magnification view of the crystalloid as seen on DIC microscopy. N, nucleus stained with Hoechst 33342. (Scale bar: 5 µm.)

DHHC10 is required for crystalloid formation. (A) WT ookinetes with crystalloids (+) under DIC microscopy (live) and after Giemsa staining (fixed). (Scale bar: 5 µm.) (B) Δdhhc10-a ookinetes show no evidence of crystalloid formation. (Scale bar: 5 µm.) (C) Longitudinal TEM section of a WT ookinete. N, nucleus; C, crystalloid surrounded by hemozoin (+). (Scale bar: 1 µm.) (D) TEM of WT crystalloid (C) reveals an ordered, honeycomb arrangement surrounded by hemozoin (+). (Scale bar: 1 µm.) (E) Apical complex of a WT ookinete with micronemes (*) and a typical arrangement of subpellicular MT, IMC, and PM. (Scale bar: 1 µm.) (F) Apical complex of Δdhhc10-b ookinetes showing no affect on micronemes (*) or MT, IMC, and PM arrangement. (Scale bar: 1 µm.) (G) Longitudinal TEM section of a Δdhhc10-b ookinete highlights the nucleus (N), but does not reveal a distinct crystalloid. Hemozoin (+) is scattered throughout the cytoplasm. (Scale bar: 1 µm.)

Absence of DHHC10 disturbs localization of crystalloid-resident protein LAP2. Live imaging of lap2::gfp (A) and Δdhhc10;lap2::gfp (B) female gametocytes shows LAP2::GFP expression throughout the gametocyte cytoplasm in both strains. Only in WT ookinetes is LAP2::GFP trafficked to crystalloids, whereas in Δdhhc10;lap2::gfp parasites, the protein remains distributed throughout the ookinete cytoplasm. DNA was stained with Hoechst 33342. (Scale bars: 5 µm.)