Activity of TEP1 and TEP3 are not protective against <i>P</i>. <i>berghei</i> sporozoites.

A. TEP1 and TEP3 expression is efficiently silenced at the time of salivary gland dissection (10 d post-dsTEP1, dsTEP3 or dsTEP1/TEP3 injections). TEP1 and TEP3 silencing was verified by the qPCR measurement. The ratio of the normalized TEP1 or TEP3 cDNA detection in dsTEP1, dsTEP3 or dsTEP1/TEP3 versus dsGFP treatments was calculated using triplicates from the same cDNA dilution. Graphs represent mean with ±SEM of the expression fold change from independent biological replicates (N). Data for qPCR analysis was analyzed by unpaired t-test (significance levels of t-test p-values: ** p-value B. TEP1, TEP3 or simultaneous TEP1/TEP3 depletion does not influence sporozoite salivary gland infection. The percentage of sporozoite-infected salivary glands (gray) or non-infected (white) in dsTEP1, dsTEP3 or dsTEP1/TEP3 mosquitoes and dsGFP injected control are shown in pie charts as the mean percentage obtained from independent number of biological replicates (N). Prevalence from each replicate was compared between the two conditions by chi-square test. All statistical differences were first tested independently within replicates (individual p-values in S2 Table), and if individual replicates showed a common trend of change, individual p-values were combined using the meta-analytical approach of Fisher (significance level of chi-square n.s. = not significant). (PDF)</p

Midgut permeabilization enables antibody access to <i>P</i>. <i>berghei</i> parasites in all midgut locations.

IFA of midguts collected 24 h post-IBM. Midguts were permeabilized and immunostained with anti-GFP conjugated antibody. GFP-expressing ookinetes (green) were also associated with anti-GFP antibody (red) which confirmed antibody accessibility to all parasites in mosquito midguts. Nuclei and actin were stained with DAPI and Phalloidin (blue). The scale bar is 10 μm. (PDF)</p

APL1C binds to extracellular <i>Plasmodium berghei</i> ookinetes <i>in vivo</i>.

A. Immunostaining of non-permeabilized P. berghei-infected mosquito midguts 24 h post-infection detects APL1C protein binding to extracellular ookinetes. Extracellular location of parasites is indicated by staining with antibody against Pys25 ookinete surface protein (yellow), APL1C binding is indicated by anti-APL1C antibody staining (red), and live parasites are indicated by expression of GFP (green). Numbered ookinetes indicate representative combinations of attributes: ookinete 1, extracellular (yellow) APL1C-positive (red) and live (green); 2, extracellular, APL1C-postive, and dead (not green); 3, the same as 2; 4, extracellular, APL1C-negative (not red), and live. Nuclei and actin were stained with DAPI and phalloidin, respectively (blue). Scale bar, 10 μm. B. The majority of extracellular ookinetes and almost all dead extracellular ookinetes are labelled by APL1C protein. All ookinetes from P. berghei-infected midguts treated as in panel A were counted and scored for attributes in three biological replicates (N, with 5–14 midguts per replicate), n indicates number of ookinetes scored. Top pie chart depicts the outcome for all extracellular (Pys25-positive) parasites (red pie slice, APL1C-positive extracellular ookinetes, green pie slice, APL1C-negative extracellular parasites), shown as the mean percentage obtained from three replicates. Bottom pie chart depicts the outcome of all dead extracellular (Pys25-positive and GFP negative) parasites (red pie slice, APL1C-positive dead parasites, yellow pie slice, APL1C-negative dead parasites). Parasite images in boxes are unmerged projections of the same numbered parasites shown in panel A. Scale bars of enlarged projection, 2 μm.</p

APL1C does not bind to ookinetes of <i>P</i>. <i>falciparum</i>.

Confocal immunostaining analysis of non-permeabilized midguts at 24 h post-infection with P. falciparum. A. The projection depicts two midgut ookinetes stained with Pfs25 protein (green), indicating that they were extracellular, but not associated with APL1C protein (red). As a positive control for APL1C detection, the IFA signal of hemolymph APL1C in the extracellular space can be seen, as in Fig 1. Scale bar, 10 μm. B. Enlarged projections depict ookinetes that are stained with Pfs25 but not APL1C, representative of the n = 47 ookinetes observed, none of which were labelled by APL1C. The bottom row depicts a diffuse APL1C signal that was occasionally observed in proximity to P. falciparum ookinetes. Nuclei and actin were stained with DAPI and phalloidin (blue). Scale bar, 2 μm.</p

V5-tagged protein constructs are secreted from the hemocyte-like 4a3A cell line.

A. Immunoblot analysis of culture medium (M) and cells (C) of 4a3A cells transfected with plasmids encoding V5-tagged RFP (RFP-V5) and V5-tagged RFP fused with the signal sequence from APL1C (sRFP-V5). Immunoblot was probed with anti-V5 antibody. B. Immunoblot analysis of culture medium of 4a3A cells transfected with plasmids encoding V5-tagged APL1C and LRIM1 under reducing (R) and non-reducing (NR) conditions with anti-V5 antibody. Estimated sizes of monomeric APL1C and LRIM1 forms including V5-tag are: 88 kDa (APL1C) and 60 kDa (LRIM1), respectively. The results indicate that both APL1C-V5 and LRIM1-V5 are secreted into the culture medium. (PDF)</p

APL1C protein is localized in hemocyte vesicles. Liposome-encapsulated clodronate injection depletes mosquito phagocytic cell markers.

A. Immunostaining analysis of perfused hemocytes and B cultured 4a3A cells indicates that APL1C protein is localized in vesicles or vesicle-like structures (red). Cells were stained with DAPI to label nuclei (blue) and phalloidin to label actin (green). Bright field indicated as BF. The scale bar is 5 μm. C. Clodronate-mediated phagocytic cell depletion was verified by the qPCR measurement of phagocytosis markers eater and nimrodB2 between mosquitoes injected with clodronate (CLD) and control empty liposomes (LP, dotted line) at 24 h post-injection. The ratio of normalized eater or nimrodB2 cDNA detection in CLD and LP treatments was calculated using triplicates from the same cDNA dilution. Graph represents mean with ±SEM of the expression fold change between CLD and LP from three biological replicates (N = 3). Data for qPCR analysis was analyzed by unpaired t-test (significance levels of t-test p-values: * p-value (PDF)</p

Bloodmeal induction of hemolymph APL1C is not dependent upon proliferation of the enteric microbiome.

A. Bacterial abundance in mosquitoes was significantly reduced after antibiotic treatment, as confirmed by qPCR quantification of 16S ribosomal gene DNA (16S rDNA) in mosquitoes exposed (+ATB) or not (control, -ATB, depicted as a dotted line) to antibiotics at 24 h post-NBM. The ratio of normalized 16S rDNA detection in “+ATB” versus “-ATB” treatments was calculated using triplicates from the same cDNA dilution. Graph represents mean with ±SEM of the fold change between +ATB and -ATB from two biological replicates (N = 2). B. Immunostaining analysis of +ATB midguts indicates that bacterial depletion did not alter APL1C protein localization on NBM midguts. Images are representative of two independent biological replicates (N = 2, 3–7 midguts per experimental point). The scale bar is 10 μm. (PDF)</p

APL1C activity in the hemocoel limits <i>P</i>. <i>berghei</i> sporozoite invasion of salivary glands.

A. APL1C protein level increases transiently in mosquitoes after either non-infective bloodmeal (NBM) or P. berghei infectious bloodmeal (IBM), and in IBM mosquitoes again during sporozoite release. APL1C levels in whole mosquitoes were determined by western blot using anti-APL1C antibody normalized to control anti-GADPH antibody. Time points correspond to presence in IBM mosquitoes of young oocysts (4 d), mature oocysts before sporozoite release (10 d), early sporozoite release (12 d) and late sporozoite release (18 d). Signals were compared to unfed (UF) controls, defined as 100% (dashed line). Graph represents mean with ±SEM of the fold-change between IBM/UF or NBM/UF, from three biological replicates (N = 3). Data were analyzed by unpaired t-test (significance levels: * p-valueB. APL1C depletion strongly increased the fraction of mosquitoes with infected salivary glands. Sporozoites were counted in the salivary glands dissected from individual mosquitoes after prior treatment with dsAPL1C or dsGFP. Each mosquito was scored as sporozoite-infected (gray) or non-infected (white) in four biological replicates (N = 4). Salivary gland infection prevalence in each replicate was compared between the two dsRNA treatment conditions by chi-square test (individual p-values in S2 Table) and were combined using the meta-analytical approach of Fisher (significance level of chi-square **** p-value C-D. APL1C protein binds sporozoites in the mosquito hemocoel. C. Sporozoites were perfused from mosquitoes at different times post-infection and analyzed by flow cytometry using anti-APL1C antibody. Graph represents the mean percentage of APL1C-positive sporozoites from at least two biological replicates, bars represent mean with ±SEM. D. APL1C binding to sporozoites was confirmed by IFA of sporozoites perfused from mosquitoes at 17 d post-infection labeled with anti-APL1C antibody. GFP-expressing sporozoites (green) were observed coated with APL1C protein (red). Nuclei were stained with DAPI (blue). Scale bar, 5 μm.</p

Activity of APL1C and LRIM1 do not directly reduce P. berghei sporozoite numbers in the mosquito hemocoel.

A. APL1C was silenced by dsRNA treatment of mosquitoes at 9 d post-IBM, prior to sporozoite release from oocysts. At 17 d post-IBM, during sporozoite release, mosquitoes were perfused individually and the number of circulating sporozoites per individual mosquito was counted by flow cytometry in mosquitoes treated with dsAPL1C or control dsGFP. Graph presents the sporozoite number in perfused individuals between dsAPL1C- and dsGFP-treated mosquitoes. Each point represents a single perfused individual, bars represent mean with ±SEM. Sample sizes (N) show the number of independent replicate experiments, (n) the total number of perfused individuals across replicates. Data were compared between the two conditions by Mann-Whitney test. All statistical differences tested independently within replicates (individual p-values in S1 Table) (significance level of Mann-Whitney n.s. = not significant). B. APL1C silencing by dsAPL1C treatment 9 d post-IBM was still efficient at the time of mosquito perfusion, 17 d post-IBM, as verified by qPCR. The ratio of the normalized APL1C cDNA detection in dsAPL1C versus dsGFP treatments was calculated using triplicates from the same cDNA dilution. Graph represents mean with ±SEM of the transcript abundance fold change between dsAPL1C and dsGFP samples from independent biological replicates (N). Data for qPCR analysis was analyzed by unpaired t-test (significance levels of t-test p-values: *** p-value C. Test of LRIM1 function for hemocoel sporozoite numbers. Description as in panel A but substituting dsLRIM1 in place of dsAPL1C. D. LRIM1 silencing by dsLRIM1 treatment 9 d post-IBM was still efficient at the time of mosquito perfusion, 17 d post-IBM, as verified by qPCR. Description as in panel B but substituting dsLRIM1 in place of dsAPL1C. (PDF)</p

APL1C antibody labelling of P. berghei sporozoites is specific to the APL1C protein.

A. Sporozoites perfused from control dsGFP-treated mosquitoes are labelled by APL1C protein. The top panel depicts merged images of representative sporozoites (scale bar 10 μm), whereas the images below show 3 channels of enlarged projections of sporozoites (scale bar 2 μm), indicated by the numbers on the merged image. B. Sporozoites perfused from dsAPL1C-treated mosquitoes are not labelled by APL1C protein. The top panel depicts merged images of representative sporozoites (scale bar 10 μm), whereas the images below show 3 channels of enlarged projections of sporozoites (scale bar 2 μm), indicated by the numbers on the merge picture. (PDF)</p