Plasmodium Sporozoites Research Articles

Long lasting insecticidal nets (LLINs) intervention impact on use, misuse and challenges in malaria and lymphatic filariasis transmission

To assess the effectiveness and misuse status of Long Lasting Insecticidal Nets (LLINs), intervention, a study was conducted in areas of malaria and lymphatic filariasis endemicity from November 2021 to August 2022. A preliminary survey preceded a cross-sectional survey of the misuse of LLINs. Considering the outcome of the former, 57 households misusing LLINs were reported. Thirty (30) cohorts each from LLIN misusing and LLIN non misusing households served as permanent cohorts for mosquito collections (60 rooms in 60 households in 3 sentinel villages). The study also investigated the status of LLIN usage and non usage. Mosquitoes caught with Aspirators and Pyrethrum Knockdown (PKD) techniques were identified using standard morphological keys. Among those who used LLIN, 68.42% used it for other purposes than vector control while 31.58% didn’t utilize it for mosquito control. About 59.65% didn’t know the effect of using it for purposes other than malaria/LF control while 40.35% were familiar. Of 8,180 endophilic mosquitoes caught and assessed for parity, infection/infectivity status, 60.07% (4,914/8,180) and 39.93% (3,266/8,180) represented LLIN misusing and LLIN non-misusing catches respectively. Overall, insignificantly higher catches were made in LLIN misusing cohorts than LLIN non misusing cohorts (63.54% versus 36.45%, 58.89% versus 41.11%, 58.86% versus 41.18%) for 2015, 2016 and 2017 respectively. Specifically, malaria/LF vector densities showed that Anopheles gambiae s.l. (82.53% versus 74.04%) was the highest followed by An. funestus s.l. (10.15% versus 12.19%) and Culex quinquefasciatus (6.82% versus 3.83%) while other species were rarely present. The physiological, infection (0.28% versus 0.49%) and infectivity (0.00% versus 0.00%) statuses with Wuchereria bancrofti were insignificant between the cohorts (p>0.05). None of the Anopheles assessed for Plasmodium sporozoite were positive for both cohorts. The percentage level of LLIN household use was 43.33% while 41.03% slept under LLIN daily, 58.97% slept sometimes. Obstacles to LLIN use included hot/heat (56.86%), dislike (21.57%), lack of protection (11.86%) and ineffectiveness (9.80%). The role of significant misuse of LLIN was highlighted by the study. The results indicated the intensity of filariasis transmission by transmitting vectors was higher than that of malaria and re-emphasis the need to promote positive attitude towards the use of LLIN for control of the two diseases.

Dihydroartemisinin suppresses the susceptibility of Anopheles stephensi to Plasmodium yoelii by activating the Toll signaling pathway

BackgroundMalaria is a serious public health concern. Artemisinin and its derivatives are first-line drugs for the treatment of Plasmodium falciparum malaria. In mammals, artemisinin exhibits potent anti-inflammatory and immunoregulatory properties. However, it is unclear whether artemisinin plays a regulatory role in the innate immunity of mosquitoes, thereby affecting the development of Plasmodium in Anopheles when artemisinin and its metabolites enter mosquitoes. This study aims to determine the effect of dihydroartemisinin (DHA), a first-generation semisynthetic derivative of artemisinin, on innate immunity and malaria vector competence of Anopheles stephensi.MethodsAnopheles stephensi was fed Plasmodium-infected mice treated with DHA via gavage, Plasmodium-infected blood containing DHA in vitro, or DHA-containing sugar, followed by Plasmodium yoelii infection. The engorged female mosquitoes were separated and dissected 8 and 17 days after infection. Plasmodium oocysts and sporozoites were counted and compared between the control and DHA-treated groups. Additionally, total RNA and proteins were extracted from engorged mosquitoes 24 and 72 h post infection (hpi). Real-time polymerase chain reaction (PCR) and western blotting were performed to detect the transcriptional levels and protein expression of immune molecules in mosquitoes. Finally, the Toll signaling pathway was inhibited via RNA interference and the infection density was analyzed to confirm the role of the Toll signaling pathway in the effect of DHA on the vector competence of mosquitoes.ResultsDHA treatment via different approaches significantly reduced the number of Plasmodium oocysts and sporozoites in mosquitoes. The transcriptional levels of anti-Plasmodium immune genes (including TEP1, LRIM1, and APL1C), Toll pathway genes (including Tube, MyD88, and Rel1), and the effector defensin 1 were upregulated by DHA treatment at 24 and 72 hpi. TEP1 and Rel1 protein expression was significantly induced under DHA treatment. However, Rel1 knockdown in DHA-treated mosquitoes abrogated DHA-mediated refractoriness to Plasmodium infection.ConclusionsDHA treatment effectively inhibited the development of P. yoelii in A. stephensi by upregulating mosquitoes’ Toll signaling pathway, thereby influencing the susceptibility of Anopheles to Plasmodium.Graphical

Bionomics and distribution of malaria vectors in Kisumu city, Western Kenya: Implications for urban malaria transmission.

Increasing urbanization in tropical Africa may create new niches for malaria vectors, potentially leading to higher disease transmission rates. Vector control efforts remain largely targeted at ecologically rural bio-complexities with multiple hosts. Understanding mosquito species composition, ecology, host diversity and biting behavior in urban areas is crucial for planning effective control. This study assessed mosquito species diversity, abundance, behavioral patterns, and Plasmodium sporozoite infection rates of Anopheles vectors along an urban-rural transect in Kisumu city, western Kenya. Indoor and outdoor host-seeking and resting adult mosquitoes were collected using Centers for Disease Control and Prevention miniature light traps (CDC-LT) and mechanical aspirators (Prokopack) along an urban-rural transect. Females Anopheles mosquitoes collected were identified using morphological taxonomic keys to species level. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were further processed using polymerase chain reaction (PCR) to identify members of each complex/group. Subsequently, sporozoite infection rates of the anopheline mosquitoes were determined using a multiplexed real-time quantitative PCR (qPCR) assay. A total of 3,394 female Anopheles mosquitoes were collected and identified. These comprised of An. gambiae s.l. (68%), An. funestus group (19.8%), An. coustani (7.8%), An. pharoensis (2.6%), An. maculipalipis (1.6%), and An. leesoni(0.2%). All six species were found in urban zone, but only three were found in peri-urban and rural sites. Overall, urban collections accounted for the majority of these collections (55.5%) of mosquitoes collected, followed by those from peri-urban (30%) and rural sites (14.5%). Species distribution across the three ecotypes showed Anopheles arabiensis was the dominant species in urban (84.3%) and peri-urban (89%) sites, while An. gambiae s.s. was predominantly found in the rural zone (60.2%) alongside An. arabiensis (39.7%). Anopheles funestus was the predominant species in peri-urban (98.4%) and rural (85.7%) areas, with An. leesoni accounted for 1.6% and 14.3%, respectively. In urban areas, all samples from the An. funestus group were identified as An. funestus s.s.. Majority (55.5%) of Anopheles mosquitoes were collected indoors, while secondary vectors were primarily caught outdoors. Overall, sporozoite rates were higher outdoors 3.5% compared to indoors 1.45% in rural areas. Specifically, sporozoite infectivity rates for An. funestus, An. gambiae s.s and An. arabiensis collected indoors in the rural zone was 2.5%, 1.4% and 1% respectively. Outdoors in rural areas, An. gambiae had a sporozoite rate of 5.3%, while An. arabiensis had a rate of 2.1%. In peri-urban areas An. gambiae had a sporozoite rate of 2.3%. No sporozoites were detected in samples from urban sites. The study highlights a shift of diversity of Anopheles species towards urban areas with increased outdoor activity, and significant outdoor malaria transmission in rural and peri-urban areas, emphasizing the need for tools targeting outdoor-biting mosquitoes. The presence of An. funestus in urban settings is of interest and highlights the critical importance of sustained entomological surveillance to inform integrated vector control and prevent future transmission risks.

Mosquito salivary apyrase regulates blood meal hemostasis and facilitates malaria parasite transmission

The evolution of hematophagy involves a series of adaptations that allow blood-feeding insects to access and consume blood efficiently while managing and circumventing the host’s hemostatic and immune responses. Mosquito, and other insects, utilize salivary proteins to regulate these responses at the bite site during and after blood feeding. We investigated the function of Anopheles gambiae salivary apyrase (AgApyrase) in regulating hemostasis in the mosquito blood meal and in Plasmodium transmission. Our results demonstrate that salivary apyrase, a known inhibitor of platelet aggregation, interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protease that degrades fibrin and facilitates Plasmodium transmission. We show that mosquitoes ingest a substantial amount of apyrase during blood feeding, which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. AgApyrase significantly enhanced Plasmodium infection in the mosquito midgut, whereas AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammalian host, underscoring the potential for strategies to prevent malaria transmission.

A Micronemal Protein, Scot1, Is Essential for Apicoplast Biogenesis and Liver Stage Development in Plasmodium berghei.

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei, we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

A geometrical theory of gliding motility based on cell shape and surface flow

Gliding motility proceeds with little changes in cell shape and often results from actively driven surface flows of adhesins binding to the extracellular environment. It allows for fast movement over surfaces or through tissue, especially for the eukaryotic parasites from the phylum apicomplexa, which includes the causative agents of the widespread diseases malaria and toxoplasmosis. We have developed a fully three-dimensional active particle theory which connects the self-organized, actively driven surface flow over a fixed cell shape to the resulting global motility patterns. Our analytical solutions and numerical simulations show that straight motion without rotation is unstable for simple shapes and that straight cell shapes tend to lead to pure rotations. This suggests that the curved shapes of Plasmodium sporozoites and Toxoplasma tachyzoites are evolutionary adaptations to avoid rotations without translation. Gliding motility is also used by certain myxo- or flavobacteria, which predominantly move on flat external surfaces and with higher control of cell surface flow through internal tracks. We extend our theory for these cases. We again find a competition between rotation and translation and predict the effect of internal track geometry on overall forward speed. While specific mechanisms might vary across species, in general, our geometrical theory predicts and explains the rotational, circular, and helical trajectories which are commonly observed for microgliders. Our theory could also be used to design synthetic microgliders.

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

Plasmodium sporozoite shows distinct motility patterns in responses to three-dimensional environments

During malaria infection, Plasmodium sporozoites, the fast-moving stage of the parasite, are injected by a mosquito into the skin of the mammalian host. In the skin, sporozoites need to migrate through the dermal tissue to enter the blood vessel. Sporozoite motility is critical for infection but not well understood. Here, we used collagen hydrogels with tunable fiber structures, as an invitro model for the skin. After injecting sporozoites into the hydrogel, we analyzed their motility in three-dimension (3D). We found that sporozoites demonstrated chiral motility, in that they mostly follow right-handed helical trajectories. In high-concentration collagen gel, sporozoites have lower instantaneous speed, but exhibit straighter tracks compared to low-concentration collagen gel, which leads to longer net displacement and faster dissemination. Taken together, our study indicates an inner mechanism for sporozoites to adapt to the environment, which could help with their successful exit from the skin tissue.

Vectorial drivers of malaria transmission in Jabi Tehnan district, Amhara Regional State, Ethiopia

Among the factors affecting the effectiveness of malaria control is poor knowledge of the entomologic drivers of the disease. We investigated anopheline populations as part of a baseline study to implement house screening of windows and doors as a supplementary malaria control tool towards elimination in Jabi Tehnan district, Amhara Regional State of Ethiopia. The samples were surveyed monthly using CDC light traps between June 2020 and May 2021. Mosquito trap density (< 3 mosquitoes/trap) was low, however, with a high overall Plasmodium sporozoite rate (9%; indoor = 4.3%, outdoor = 13.1%) comprising P. falciparum (88.9%) and P. vivax (11.1%). Anopheles gambiae s.l., mostly An. arabiensis, comprised > 80% of total anopheline captures and contributed ~ 42% of Plasmodium-infected mosquitoes. On the other hand, morphologically scored Anopheles funestus s.l., constituting about 6% of anopheline collections, accounted for 50% of sporozoite-infected mosquitoes. Most of the infected An. funestus s.l. specimens (86.7%) were grouped with previously unknown or undescribed Anopheles species previously implicated as a cryptic malaria vector in the western Kenyan highlands, confirming its wider geographic distribution in eastern Africa. Other species with Plasmodium infection included An. longipalpis C, An. theileri, An. demillioni, and An. nili. Cumulatively, 77.8% of the infected mosquitoes occurred outdoors. These results suggest efficient malaria parasite transmission despite the low vector densities, which has implications for effective endpoint indicators to monitor malaria control progress. Additionally, the largely outdoor infection and discovery of previously unknown and cryptic vectors suggest an increased risk of residual malaria transmission and, thus, a constraint on effective malaria prevention and control.

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Reagent-free detection of Plasmodium falciparum malaria infections in field-collected mosquitoes using mid-infrared spectroscopy and machine learning.

Field-derived metrics are critical for effective control of malaria, particularly in sub-Saharan Africa where the disease kills over half a million people yearly. One key metric is entomological inoculation rate, a direct measure of transmission intensities, computed as a product of human biting rates and prevalence of Plasmodium sporozoites in mosquitoes. Unfortunately, current methods for identifying infectious mosquitoes are laborious, time-consuming, and may require expensive reagents that are not always readily available. Here, we demonstrate the first field-application of mid-infrared spectroscopy and machine learning (MIRS-ML) to swiftly and accurately detect Plasmodium falciparum sporozoites in wild-caught Anopheles funestus, a major Afro-tropical malaria vector, without requiring any laboratory reagents. We collected 7178 female An. funestus from rural Tanzanian households using CDC-light traps, then desiccated and scanned their heads and thoraces using an FT-IR spectrometer. The sporozoite infections were confirmed using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), to establish references for training supervised algorithms. The XGBoost model was used to detect sporozoite-infectious specimen, accurately predicting ELISA and PCR outcomes with 92% and 93% accuracies respectively. These findings suggest that MIRS-ML can rapidly detect P. falciparum in field-collectedmosquitoes, with potential for enhancing surveillance in malaria-endemic regions. The technique is both fast, scanning 60-100 mosquitoes per hour, and cost-efficient, requiring no biochemical reactions and therefore no reagents. Given its previously proven capability in monitoring key entomological indicators like mosquito age, human blood index, and identities of vector species, we conclude that MIRS-ML could constitute a low-cost multi-functional toolkit for monitoring malaria risk and evaluating interventions.

The novel Plasmodium berghei protein S14 is essential for sporozoite gliding motility and infectivity.

Plasmodium sporozoites are the infective forms of the malaria parasite in the mosquito and vertebrate host. Gliding motility allows sporozoites to migrate and invade mosquito salivary glands and mammalian hosts. Motility and invasion are powered by an actin-myosin motor complex linked to the glideosome, which contains glideosome-associated proteins (GAPs), MyoA and the myosin A tail-interacting protein (MTIP). However, the role of several proteins involved in gliding motility remains unknown. We identified that the S14 gene is upregulated in sporozoite from transcriptome data of Plasmodium yoelii and further confirmed its transcription in P. berghei sporozoites using real-time PCR. C-terminal 3×HA-mCherry tagging revealed that S14 is expressed and localized on the inner membrane complex of the sporozoites. We disrupted S14 in P. berghei and demonstrated that it is essential for sporozoite gliding motility, and salivary gland and hepatocyte invasion. The gliding and invasion-deficient S14 knockout sporozoites showed normal expression and organization of inner membrane complex and surface proteins. Taken together, our data show that S14 plays a role in the function of the glideosome and is essential for malaria transmission.

Effect of spatiotemporal variables on abundance, biting activity and parity of Nyssorhynchus darlingi (Diptera: Culicidae) in peri-Iquitos, Peru

BackgroundIn malaria endemic regions of the Peruvian Amazon, rainfall together with river level and breeding site availability drive fluctuating vector mosquito abundance and human malaria cases, leading to temporal heterogeneity. The main variables influencing spatial transmission include location of communities, mosquito behaviour, land use/land cover, and human ecology/behaviour. The main objective was to evaluate seasonal and microgeographic biting behaviour of the malaria vector Nyssorhynchus (or Anopheles) darlingi in Amazonian Peru and to investigate effects of seasonality on malaria transmission.MethodsWe captured mosquitoes from 18:00 to 06:00 h using Human Landing Catch in two riverine (Lupuna, Santa Emilia) and two highway (El Triunfo, Nuevo Horizonte) communities indoors and outdoors from 8 houses per community, during the dry and rainy seasons from February 2016 to January 2017. We then estimated parity rate, daily survival and age of a portion of each collection of Ny. darlingi. All collected specimens of Ny. darlingi were tested for the presence of Plasmodium vivax or Plasmodium falciparum sporozoites using real-time PCR targeting the small subunit of the 18S rRNA.ResultsAbundance of Ny. darlingi varied across village, season, and biting behaviour (indoor vs outdoor), and was highly significant between rainy and dry seasons (p < 0.0001). Biting patterns differed, although not significantly, and persisted regardless of season, with peaks in highway communities at ~ 20:00 h in contrast to biting throughout the night (i.e., 18:00–06:00) in riverine communities. Of 3721 Ny. darlingi tested for Plasmodium, 23 (0.62%) were infected. We detected Plasmodium-infected Ny. darlingi in both community types and most (20/23) were captured outdoors during the rainy season; 17/23 before midnight. Seventeen Ny. darlingi were infected with P. vivax, and 6 with P. falciparum. No infected Ny. darlingi were captured during the dry season. Significantly higher rates of parity were detected in Ny. darlingi during the rainy season (average 64.69%) versus the dry season (average 36.91%) and by community, Lupuna, a riverine village, had the highest proportion of parous to nulliparous females during the rainy season.ConclusionsThese data add a seasonal dimension to malaria transmission in peri-Iquitos, providing more evidence that, at least locally, the greatest risk of malaria transmission is outdoors during the rainy season mainly before midnight, irrespective of whether the community was located adjacent to the highway or along the river.

Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia

BackgroundMalaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia.MethodsEntomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households’ exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source.ResultsCatches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75–100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9–100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax).ConclusionsThis study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission.Graphical

Killing of Plasmodium Sporozoites by Basic Amphipathic α-Helical Fusion Peptides.

Membranolytic molecules constitute the first line of innate immune defense against pathogenic microorganisms. Plasmodium sporozoites are potentially exposed to these cytotoxic molecules in the hemolymph and salivary glands of mosquitoes, as well as in the skin, blood, and liver of the mammalian host. Here, we show that sporozoites are resistant to bacteriolytic concentration of cecropin B, a cationic amphipathic antimicrobial insect peptide. Intriguingly, anti-tumoral cell-penetrating peptides derived from the anti-apoptotic protein AAC11 killed P. berghei and P. falciparum sporozoites. Using dynamic imaging, we demonstrated that the most cytotoxic peptide, called RT39, did not significantly inhibit the sporozoite motility until the occurrence of a fast permeabilization of the parasite membrane by the peptide. Concomitantly, the cytosolic fluorescent protein constitutively expressed by sporozoites leaked from the treated parasite body while To-Pro 3 and FITC-labeled RT39 internalized, respectively, binding to the nucleic acids and membranes of sporozoites. This led to an increase in the parasite granularity as assessed by flow cytometry. Most permeabilization events started at the parasite's posterior end, resulting in the appearance of a fluorescent dot in the anterior part of sporozoites. Understanding and exploiting the susceptibility of sporozoites and other plasmodial stages to membranolytic molecules might foster strategies to eliminate the parasite and block its transmission.

Overcoming the egress block of Plasmodium sporozoites expressing fluorescently tagged circumsporozoite protein.

Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI-anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full-length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP-GFP. Replacing the N-terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP-GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP-GFP expressing parasites that are blocked in egress with wild-type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP-GFP. Expressing CSP-GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP-GFP without disruption of oocyst egress and salivary gland invasion.

The Anopheles leucine-rich repeat protein APL1C is a pathogen binding factor recognizing Plasmodium ookinetes and sporozoites.

Leucine-rich repeat (LRR) proteins are commonly involved in innate immunity of animals and plants, including for pattern recognition of pathogen-derived elicitors. The Anopheles secreted LRR proteins APL1C and LRIM1 are required for malaria ookinete killing in conjunction with the complement-like TEP1 protein. However, the mechanism of parasite immune recognition by the mosquito remains unclear, although it is known that TEP1 lacks inherent binding specificity. Here, we find that APL1C and LRIM1 bind specifically to Plasmodium berghei ookinetes, even after depletion of TEP1 transcript and protein, consistent with a role for the LRR proteins in pathogen recognition. Moreover, APL1C does not bind to ookinetes of the human malaria parasite Plasmodium falciparum, and is not required for killing of this parasite, which correlates LRR binding specificity and immune protection. Most of the live P. berghei ookinetes that migrated into the extracellular space exposed to mosquito hemolymph, and almost all dead ookinetes, are bound by APL1C, thus associating LRR protein binding with parasite killing. We also find that APL1C binds to the surface of P. berghei sporozoites released from oocysts into the mosquito hemocoel and forms a potent barrier limiting salivary gland invasion and mosquito infectivity. Pathogen binding by APL1C provides the first functional explanation for the long-known requirement of APL1C for P. berghei ookinete killing in the mosquito midgut. We propose that secreted mosquito LRR proteins are required for pathogen discrimination and orientation of immune effector activity, potentially as functional counterparts of the immunoglobulin-based receptors used by vertebrates for antigen recognition.

Zeb2 drives the formation of CD11c+ atypical B cells to sustain germinal centers that control persistent infection.

CD11c+ atypical B cells (ABCs) are an alternative memory B cell lineage associated with immunization, infection, and autoimmunity. However, the factors that drive the transcriptional program of ABCs have not been identified, and the function of this population remains incompletely understood. Here, we identified candidate transcription factors associated with the ABC population based on a human tonsillar B cell single-cell dataset. We identified CD11c+ B cells in mice with a similar transcriptomic signature to human ABCs, and using an optimized CRISPR-Cas9 knockdown screen, we observed that loss of zinc finger E-box binding homeobox 2 (Zeb2) impaired ABC formation. Furthermore, ZEB2 haplo-insufficient Mowat-Wilson syndrome (MWS) patients have decreased circulating ABCs in the blood. In Cd23Cre/+Zeb2fl/fl mice with impaired ABC formation, ABCs were dispensable for efficient humoral responses after Plasmodium sporozoite immunization but were required to control recrudescent blood-stage malaria. Immune phenotyping revealed that ABCs drive optimal T follicular helper (TFH) cell formation and germinal center (GC) responses and they reside at the red/white pulp border, likely permitting better access to pathogen antigens for presentation. Collectively, our study shows that ABC formation is dependent on Zeb2, and these cells can limit recrudescent infection by sustaining GC reactions.

Longitudinal analysis of antibody responses to Plasmodium vivax sporozoite antigens following natural infection.

P. vivax malaria is a major global health burden hindering social and economic development throughout many tropical and sub-tropical countries. Pre-erythrocytic (PE) vaccines emerge as an attractive approach for the control and elimination of malaria infection. Therefore, evaluating the magnitude, longevity and prevalence of naturally acquired IgG antibody responses against PE candidate antigens is useful for vaccine design. The antigenicity of five recombinant PE antigens (PvCSP-VK210, PvSSP3, PvM2-MAEBL, PvCelTOS and PvSPECT1) was evaluated in plasma samples from individuals residing in low transmission areas in Thailand (Ranong and Chumphon Provinces). The samples were collected at the time of acute vivax malaria and 90, 270 and 360 days later. The prevalence, magnitude and longevity of total IgG and IgG subclasses were determined for each antigen using the longitudinal data. Our results showed that seropositivity of all tested PE antigens was detected during infection in at least some subjects; anti-PvCSP-VK210 and anti-PvCelTOS antibodies were the most frequent. Titers of these antibodies declined during the year of follow up, but notably seropositivity persisted. Among seropositive subjects at post-infection, high number of subjects possessed antibodies against PvCSP-VK210. Anti-PvSSP3 antibody responses had the longest half-life. IgG subclass profiling showed that the predominant subclasses were IgG1 and IgG3 (cytophilic antibodies), tending to remain detectable for at least 360 days after infection. The present study demonstrated the magnitude and longevity of serological responses to multiple PE antigens of P. vivax after natural infection. This knowledge could contribute to the design of an effective P. vivax vaccine.

Revisiting the Plasmodium sporozoite inoculum and elucidating the efficiency with which malaria parasites progress through the mosquito

Malaria is initiated when infected anopheline mosquitoes inoculate sporozoites as they probe for blood. It is thought that all infected mosquitoes are equivalent in terms of their infectious potential, with parasite burden having no role in transmission success. In this study, using mosquitoes harboring the entire range of salivary gland sporozoite loads observed in the field, we demonstrate a strong and highly significant correlation between mosquito parasite burden and inoculum size. We then link the inoculum data to oocyst counts, the most commonly-used metric to assess mosquito infection in the field, and determine the efficiency with which oocyst sporozoites enter mosquito salivary glands. Taken together our data support the conclusion that mosquitoes with higher parasite burdens are more likely to initiate infection and contribute to onward transmission. Overall these data may account for some of the unexplained heterogeneity in transmission and enable more precise benchmarks for transmission-blocking interventions.