Parasites, mice, and mosquitoes
For P. berghei infections, 6- to 8-week-old male BALB/c mice (SLC, Japan) were infected with either wild type (WT) P. berghei (ANKA strain) or P. berghei (ANKA strain) which constitutively expresses GFP (Ishino et al., 2006).
Anopheles stephensi (STE2 strain) mosquitoes were maintained at 27°C and 80% relative humidity with a 14/10 h light/dark cycle in an insectary and fed 10% (w/v) sucrose solution.
For the mosquito infection experiments, P. berghei-infected mice having 10 exflagellations per 104 RBCs or more were used. In the mosquito-to-mouse infection experiment, we used mosquitoes infected with WT and knockout (KO) parasites (25 days after the mosquitoes had ingested the infected blood meal).
Construction of OSCP-KO parasites
Procedures were as previously reported [23, 25], using PCR amplification with primers OSCP-F1 (5’ -CAA AAC AAT GTG GAT TTG TG -3’) and OSCP-R1 (5’ -GGT TTT CAT TTT CCC TAA AAT C -3’) and P. berghei genomic DNA as a template. The gene was cloned into the pCR-BluntII-TOPO vector (Thermo Fisher Scientific), resulting in the plasmid pOSCP. Subsequently, pOSCP was digested using Acc I. The digested pOSCP was inserted into the hdhfr expression cassette . pOSCP (10 μg) was linearized with Xho I and electroporated into cultured P. berghei schizonts using Nucleofector II (Lonza, Basel, Switzerland). Transfected parasites were intravenously injected into male BALB/c mice that were then treated with pyrimethamine (70 μg/ml) 24 h later via drinking water.
PCR with the following primer combinations were performed to detect the presence of recombinant parasites. T1: OSCP-F2 (5’ -CCA TAC CTT CAA GAT TAG ATG AC -3’) with hDHFR-shDR (5’ -CTG TTA TAA TTA TGT TGT CTC TTC -3’), T2: hDHFR-shDF (5’ -CGA AAA GAA TTA AGC TTA ACT C -3’) with OSCP-R3 (5’ -GCA GAT CCG TCC GTT TAA C –3’), and T3: OSCP-F2 with OSCP-R2.
To analyze OSCP expression during oocyst stage, using the Trizol reagent (Thermo Fisher Scientific, MA, USA), total RNA was isolated from the mosquito at 10 days after infection. cDNA was synthesized using the ReverTra Ace kit (Toyobo, Osaka, Japan). PCR was performed using following primers OSCP-F3 (5’ -TCG AGA TGG ATG CAA AGA CTA GCA G-3’) and OSCP-R3 (5’ -GAT TCA CTG AAG GCT CAG GTT TAC C -3’).
Anti-OSCP antibody preparation and purification
Rabbit polyclonal antibodies were raised against the following OSCP protein domains: α1 (amino acids 926–939) and α2 (mixed amino acids 1789–1802 and 3103–3116) (Eurofins Genomics Inc., Tokyo, Japan) (Figure 1a). Antibodies were purified by saturated ammonium sulfate solution and Ab-Rapid SPiN EX (ProteNova, Japan). Ab-Rapid SPiN EX was performed following the manual until equilibration occurred. The purified antibodies were designated as anti-OSCP α1 and α2 antibodies. As a control, we also prepared non-immune rabbit antibodies by processing non-immune rabbit blood serum in the same manner.
Blood-stage parasite and exflagellation analysis
Parasitemia and gametocyte sex ratio were determined by preparing blood smear specimens from infected mice, fixing these with 100% methanol and then performing Giemsa staining. Exflagellation of male gametocytes was quantified as previously described [22, 27]. Briefly, 2 μl of gametocyte-infected blood was obtained from the tail vein and mixed immediately with 38 μl of the complete ookinete culture medium. The mixture was placed under a coverslip at RT, and 5 min later, exflagellation centers were counted over the next 10 min using a phase contrast microscope. The number of exflagellation centers/104 RBCs was measured per 8 μl using a Thomas hemocytometer (Nippon Rinsho Kikai Kogyo Co., Ltd., Japan).
Ookinete culture and purification
Blood from mice with exflagellation of 10/104 RBCs was obtained by heart puncture. Ten volumes of OKM was added to the blood and cultured for 24 h at 19°C. The size and number of ookinetes was measured by taking a smear from the culture solution. Ookinetes were purified using a MidiMACS separator system (Miltenyi Biotec, Germany) as previously described . A total of 3 ml culture was passed through three times before removing the column from the magnet. The ookinetes were recovered by passing 5 ml of OKM through the column. The purified ookinetes were centrifuged at 1,000 ×g 10 min at 4°C and then washed three times with PBS.
Immunofluorescent assays (IFAs)
Cultured ookinetes were placed for 30 min at 4°C on a MAS-coated adhesive slide glass (Matsunami, Japan), fixed with 4% paraformaldehyde (PFA)/PBS at room temperature for 10 min, and then washed 3 times with PBS for 5 min each time. After incubating for 10 min with 0.1 M glycine (Wako, Japan), they were washed 3 times with PBS for 5 min each time. Ookinetes were permeabilized for 10 min with 0.2% TritonX-100/PBS and washed with PBS for 10 min. No TritonX-100 was added when observing OSCP surface localization. Blocking was performed for 1 h at room temperature using 1% BSA/PBS. The primary antibody, anti-OSCP serum (α1 to α2 ratio 1:1) diluted 800-fold with PBS, was incubated for 1 h at room temperature and then washed 3 times with PBS for 5 min each time. Incubation with the secondary antibody, Alexa Fluor® 488 goat anti-rabbit IgG (H + L) (Thermo Fisher) diluted 1000-fold with PBS, was for 1 h at room temperature followed by three washes with PBS for 5 min each time. The parasites were mounted in SlowFade®Diamond Antifade Mountant with DAPI (Molecular probes, U.S.) and observed using an Eclipse E600 (Nikon, Japan) fluorescence microscope. The oocysts were observed by dissecting mosquitoes on day 15 after ingesting an infected blood meal, and staining was performed. Methanol fixation was performed, and after air-drying, the oocysts were treated with Image-iT FX Signal Enhancer solution (Thermo Fisher) for 30 min at 37°C. After PBS washing, 400-fold-diluted anti-OSCP serum was added as the primary antibody and allowed to react for 2 h at 4°C. After 3 PBS washes, Alexa Fluor® 488 goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher) was added and allowed to react for 1 h at 4°C. After 3 PBS washes, 1000-fold-diluted anti-PbCap380 rabbit serum was added as the primary antibody and allowed to react for 2 h at 4°C. As the secondary antibody, Alexa Fluor® 568 goat anti-rabbit IgG (H+L) (Thermo Fisher) was added and allowed to react for 1 h at 4°C. After 3 PBS washes and mounting, observation was performed with an Eclipse E600 (Nikon) fluorescence microscope.
Ookinete movement was investigating by mixing ookinetes with Matrigel (Corning, US) and then measuring displacement speed [9, 29]. Purified ookinete suspension and Matrigel were mixed in equal amounts, and then 15 µl drops were applied to glass slides. Glass covers (18×24 mm) were placed on top and the specimens were left to stand for 20 min on a ThermoPlate (NHP-45N; Nissin, Japan) at 19°C. Next, a digital microscope (KH-8700; Hirox, Japan) was used to observe ookinete movement for 2 min. When imaging 10-minute movement trajectories, WT and KO parasite were incubated for 30 min at 4°C with MitoBright LT Red (Dojindo, Japan) diluted 1000-fold with PBS. Centrifugation was performed at 1,000 ×g for 10 min at 4°C and then washed with PBS. This process was performed twice. Then, as stated above, the specimens were mixed with Matrigel and left to stand. They were then observed and imaged with IX83 CellSens (Olympus, Japan). In the antibody reaction experiment, antibodies diluted with PBS were added to the purified ookinete suspension and mixed with an identical amount of Matrigel before undergoing the same process. Non-immune rabbit antibodies were used as the negative control.
Oocyst number and size
Midgut oocyst numbers were counted using a Leica M205 FA (Leica, Germany) after staining the infected midguts with 0.5% mercurochrome/distilled water for 5 min and then washing with PBS for 5 min (Usui et al., 2011). The midguts were also imaged with Leica Application Suite X (Leica, Germany). We randomly selected 60 oocysts from the imaged midguts and used Image J to measure oocyst size.
The ratio of normal to deformed oocysts was observed in mosquitoes on days 6 to 7 after the blood meal. Oocysts with an internal vacuole were considered deformed.
Transmission-blocking (TB) experiment using antibodies
BALB/c mice were infected with GFP parasites by intraperitoneal injection of 106 parasites. Mice that exhibited exflagellation of 10/104 RBCs or more 5 days after infection were used for the experiments. In total, 50 female mosquitoes (Pre-group) were allowed to feed on the infected mouse for 15 min. This was followed by intravenous injection of 200 µg of anti-OSCP antibodies (α1:α2 = 1:1). After 5 min, another 50 mosquitoes (Post group) were allowed to feed for 15 min on the same mouse. Oocysts were counted 2 or 10 days later. For the TB experiment assayed 2 days after the blood meal, oocyst numbers were counted again after washing the guts with PBS for 1 min to remove any attached ookinetes.
Transmission electron microscopy
On days 17 after the infected blood meal, mosquito midguts were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.05 M sodium cacodylate buffer (pH 7.4) at 4°C for 2 h. After rinsing, samples were post-fixed at RT in buffered 1% osmium tetroxide for 2 h and then dehydrated in ethanol and propylene oxide series and embedded in epoxy resin. Ultrathin sections were cut using an Ultracut N (Reichert-Nissei, Tokyo, Japan) and stained using uranyl acetate followed by lead citrate. Sections were examined using an H-7650 transmission electron microscope (Hitachi Ltd., Tokyo, Japan).
Welch’s t-test was used to perform comparisons between groups (parasitemia, gametocytemia, number of ookinete, ookinete speed, oocyst size). The Mann–Whitney U test was used to compare the number of oocysts/midgut. All analyses were performed with GraphPad Prism software (GraphPad, San Diego, CA, U.S.).