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 Plasmodium 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 the presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for 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.

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 [ 3 H]-palmitic acid and [ 3 H]-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.

Recurrence patterns and evolution of submicroscopic and asymptomatic Plasmodium vivax infections in malaria-endemic areas of the Peruvian Amazon.

BACKGROUND: In the Peruvian Amazon, Plasmodium vivax malaria transmission is maintained due to the high frequency of recurrences. By understanding the recurrence rates of submicroscopic and asymptomatic cases, we can develop informed strategies to prevent transmission more efficiently and disrupt the silent transmission cycle. METHODS: A three-year, population-based cohort study was conducted in two sites, Cahuide and Lupuna, within the Loreto region in Peru from 2013 to 2015. The study included 385 individuals and aimed to examine the temporal dynamics of malaria recurrences and their impact on transmission and control. RESULTS: Individuals from Lupuna presented a higher risk of P. vivax infections compared to Cahuide, where most recurrences were asymptomatic and submicroscopic. It is estimated that a great proportion of these recurrences were due to relapses in both communities. The application of molecular diagnostic method proved to be significantly more effective, detecting 2.3 times more episodes during the follow-up (PCR, 1068; microscopy, 467). PCR identified recurrences significantly earlier, at 151 days after an initial infection, compared to microscopy, which detected them on average after 365 days. Community, occupation and previous malaria infections were factors associated with recurrences. Finally, potential infection evolution scenarios were described where one frequent scenario involved the transition from symptomatic to asymptomatic infections with a mean evolution time of 240 days. CONCLUSIONS: This study explores the contrast in malaria recurrence risk among individuals from two endemic settings, a consequence of prolonged exposure to the parasite. Through the analysis of the evolution scenarios of P. vivax recurrences, it is possible to have a more complete vision of how the transmission pattern changes over time and is conditioned by different factors.

Epidemiological characteristics of P. vivax asymptomatic infections in the Peruvian Amazon.

Introduction: Herein, we tested the hypothesis that Asymptomatic P. vivax (Pv) infected individuals (Asym) feature different epidemiological, clinical and biochemical characteristics, as well as hematological parameters, potentially predictive of clinical immunity in comparison to symptomatic Pv infected individuals (Sym). Methodology: Between 2018 - 2021, we conducted 11 population screenings (PS, Day 0 (D0)) in 13 different riverine communities around Iquitos city, in the Peruvian Amazon, to identify Pv Sym and Asym individuals. A group of these individuals agreed to participate in a nested case - control study to evaluate biochemical and hematological parameters. Pv Asym individuals did not present common malaria symptoms (fever, headache, and chills), had a positive/negative microscopy result, a positive qPCR result, reported no history of antimalarial treatment during the last month, and were followed-up weekly until Day 21 (D21). Control individuals, had a negative malaria microscopy and qPCR result, no history of antimalarial treatment or malaria infections during the last three years, and no history of comorbidities or chronic infections. Results: From the 2159 individuals screened during PS, data revealed a low but heterogeneous Pv prevalence across the communities (11.4%), where most infections were Asym (66.7%) and submicroscopic (82.9%). A total of 29 Asym, 49 Sym, and 30 control individuals participated in the nested case - control study (n=78). Ten of the individuals that were initially Asym at D0, experienced malaria symptoms during follow up and therefore, were included in the Sym group. 29 individuals remained Asym throughout all follow-ups. High levels of eosinophils were found in Asym individuals in comparison to Sym and controls. Conclusion: For the first-time, key epidemiological, hematological, and biochemical features are reported from Pv Asym infections from the Peruvian Amazon. These results should be considered for the design and reshaping of malaria control measures as the country moves toward malaria elimination.

Insights into Plasmodium vivax Asymptomatic Malaria Infections and Direct Skin-Feeding Assays to Assess Onward Malaria Transmission in the Amazon.

Understanding the reservoir and infectivity of Plasmodium gametocytes to vector mosquitoes is crucial to align strategies aimed at malaria transmission elimination. Yet, experimental information is scarce regarding the infectivity of Plasmodium vivax for mosquitoes in diverse epidemiological settings where the proportion of asymptomatically infected individuals varies at a microgeographic scale. We measured the transmissibility of clinical and subclinical P. vivax malaria parasite carriers to the major mosquito vector in the Amazon Basin, Nyssorhynchus darlingi (formerly Anopheles). A total of 105 participants with natural P. vivax malaria infection were recruited from a cohort study in Loreto Department, Peruvian Amazon. Four of 18 asymptomatic individuals with P. vivax positivity by blood smear infected colony-grown Ny. darlingi (22%), with 2.6% (19 of 728) mosquitoes infected. In contrast, 77% (44/57) of symptomatic participants were infectious to mosquitoes with 51% (890 of 1,753) mosquitoes infected. Infection intensity was greater in symptomatic infections (mean, 17.8 oocysts/mosquito) compared with asymptomatic infections (mean, 0.28 oocysts/mosquito), attributed to parasitemia/gametocytemia level. Paired experiments (N = 27) using direct skin-feeding assays and direct membrane mosquito-feeding assays showed that infectivity to mosquitoes was similar for both methods. Longitudinal studies with longer follow-up of symptomatic and asymptomatic parasite infections are needed to determine the natural variations of disease transmissibility.

Malaria Resilience in South America: Epidemiology, Vector Biology, and Immunology Insights from the Amazonian International Center of Excellence in Malaria Research Network in Peru and Brazil.

The 1990s saw the rapid reemergence of malaria in Amazonia, where it remains an important public health priority in South America. The Amazonian International Center of Excellence in Malaria Research (ICEMR) was designed to take a multidisciplinary approach toward identifying novel malaria control and elimination strategies. Based on geographically and epidemiologically distinct sites in the Northeastern Peruvian and Western Brazilian Amazon regions, synergistic projects integrate malaria epidemiology, vector biology, and immunology. The Amazonian ICEMR's overarching goal is to understand how human behavior and other sociodemographic features of human reservoirs of transmission-predominantly asymptomatically parasitemic people-interact with the major Amazonian malaria vector, Nyssorhynchus (formerly Anopheles) darlingi, and with human immune responses to maintain malaria resilience and continued endemicity in a hypoendemic setting. Here, we will review Amazonian ICEMR's achievements on the synergies among malaria epidemiology, Plasmodium-vector interactions, and immune response, and how those provide a roadmap for further research, and, most importantly, point toward how to achieve malaria control and elimination in the Americas.

Harmonic convergence coordinates swarm mating by enhancing mate detection in the malaria mosquito Anopheles gambiae.

The mosquito Anopheles gambiae is a major African malaria vector, transmitting parasites responsible for significant mortality and disease burden. Although flight acoustics are essential to mosquito mating and present promising alternatives to insecticide-based vector control strategies, there is limited data on mosquito flight tones during swarming. Here, for the first time, we present detailed analyses of free-flying male and female An. gambiae flight tones and their harmonization (harmonic convergence) over a complete swarm sequence. Audio analysis of single-sex swarms showed synchronized elevation of male and female flight tones during swarming. Analysis of mixed-sex swarms revealed additional 50 Hz increases in male and female flight tones due to mating activity. Furthermore, harmonic differences between male and female swarm tones in mixed-sex swarms and in single-sex male swarms with artificial female swarm audio playback indicate that frequency differences of approximately 50 Hz or less at the male second and female third harmonics (M2:F3) are maintained both before and during mating interactions. This harmonization likely coordinates male scramble competition by maintaining ideal acoustic recognition within mating pairs while acoustically masking phonotactic responses of nearby swarming males to mating females. These findings advance our knowledge of mosquito swarm acoustics and provide vital information for reproductive control strategies.

The impact of mating and sugar feeding on blood-feeding physiology and behavior in the arbovirus vector mosquito Aedes aegypti.

BACKGROUND: Aedes aegypti mosquitoes are globally distributed vectors of viruses that impact the health of hundreds of millions of people annually. Mating and blood feeding represent fundamental aspects of mosquito life history that carry important implications for vectorial capacity and for control strategies. Females transmit pathogens to vertebrate hosts and obtain essential nutrients for eggs during blood feeding. Further, because host-seeking Ae. aegypti females mate with males swarming near hosts, biological crosstalk between these behaviors could be important. Although mating influences nutritional intake in other insects, prior studies examining mating effects on mosquito blood feeding have yielded conflicting results. METHODOLOGY/PRINCIPAL FINDINGS: To resolve these discrepancies, we examined blood-feeding physiology and behavior in virgin and mated females and in virgins injected with male accessory gland extracts (MAG), which induce post-mating changes in female behavior. We controlled adult nutritional status prior to blood feeding by using water- and sugar-fed controls. Our data show that neither mating nor injection with MAG affect Ae. aegypti blood intake, digestion, or feeding avidity for an initial blood meal. However, sugar feeding, a common supplement in laboratory settings but relatively rare in nature, significantly affected all aspects of feeding and may have contributed to conflicting results among previous studies. Further, mating, MAG injection, and sugar intake induced declines in subsequent feedings after an initial blood meal, correlating with egg production and laying. Taking our evaluation to the field, virgin and mated mosquitoes collected in Colombia were equally likely to contain blood at the time of collection. CONCLUSIONS/SIGNIFICANCE: Mating, MAG, and sugar feeding impact a mosquito's estimated ability to transmit pathogens through both direct and indirect effects on multiple aspects of mosquito biology. Our results highlight the need to consider natural mosquito ecology, including diet, when assessing their physiology and behavior in the laboratory.