Considerations for first field trials of low-threshold gene drive for malaria vector control.

Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a 'causal pathway' would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

Immune interactions between mosquitoes and microbes during midgut colonization.

Mosquitoes encounter diverse microbes during their lifetime, including symbiotic bacteria, shaping their midgut ecosystem. The organization of the midgut supports microbiota persistence while defending against potential pathogens. The influx of nutrients during blood feeding triggers bacterial proliferation, challenging host homeostasis. Immune responses, aimed at controlling bacterial overgrowth, impact blood-borne pathogens such as malaria parasites. However, parasites deploy evasion strategies against mosquito immunity. Leveraging these mechanisms could help engineer malaria-resistant mosquitoes, offering a transformative tool for malaria elimination.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center in 2023.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) is a Bioinformatics Resource Center funded by the National Institutes of Health with additional funding from the Wellcome Trust. VEuPathDB supports >600 organisms that comprise invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Since 2004, VEuPathDB has analyzed omics data from the public domain using contemporary bioinformatic workflows, including orthology predictions via OrthoMCL, and integrated the analysis results with analysis tools, visualizations, and advanced search capabilities. The unique data mining platform coupled with >3000 pre-analyzed data sets facilitates the exploration of pertinent omics data in support of hypothesis driven research. Comparisons are easily made across data sets, data types and organisms. A Galaxy workspace offers the opportunity for the analysis of private large-scale datasets and for porting to VEuPathDB for comparisons with integrated data. The MapVEu tool provides a platform for exploration of spatially resolved data such as vector surveillance and insecticide resistance monitoring. To address the growing body of omics data and advances in laboratory techniques, VEuPathDB has added several new data types, searches and features, improved the Galaxy workspace environment, redesigned the MapVEu interface and updated the infrastructure to accommodate these changes.

Intracellular Plasmodium aquaporin 2 is important for sporozoite production in the mosquito vector and malaria transmission.

Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of Plasmodium development in the mosquito vector. Disruption of AQP2 in the human parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in P. berghei, combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.

Identification of genes required for Plasmodium gametocyte-to-sporozoite development in the mosquito vector.

Malaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, the complex regulation of gene expression and genotype-phenotype associations in the mosquito vector, along with sexual reproduction, have hindered the development of screens in this critical part of the parasite life cycle. To address this, we developed a genetic approach in the rodent parasite Plasmodium berghei that, in combination with barcode sequencing, circumvents the fertilization roadblock and enables screening for gametocyte-expressed genes required for parasite infection of the mosquito Anopheles coluzzii. Our results confirm previous findings, validating our approach for scaling up, and identify genes necessary for mosquito midgut infection, oocyst development, and salivary gland infection. These findings can aid efforts to study malaria transmission biology and to develop interventions for controlling disease transmission.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles gambiae, Giles, 1902, Ifakara strain.

We present a genome assembly from an individual female Anopheles gambiae (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), Ifakara strain. The genome sequence is 264 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

Climate change and human health in the Eastern Mediterranean and Middle East: Literature review, research priorities and policy suggestions.

Human health is linked to climatic factors in complex ways, and climate change can have profound direct and indirect impacts on the health status of any given region. Susceptibility to climate change is modulated by biological, ecological and socio-political factors such as age, gender, geographic location, socio-economic status, occupation, health status and housing conditions, among other. In the Eastern Mediterranean and Middle East (EMME), climatic factors known to affect human health include extreme heat, water shortages and air pollution. Furthermore, the epidemiology of vector-borne diseases (VBDs) and the health consequences of population displacement are also influenced by climate change in this region. To inform future policies for adaptation and mitigation measures, and based on an extensive review of the available knowledge, we recommend several research priorities for the region. These include the generation of more empirical evidence on exposure-response functions involving climate change and specific health outcomes, the development of appropriate methodologies to evaluate the physical and psychological effects of climate change on vulnerable populations, determining how climate change alters the ecological determinants of human health, improving our understanding of the effects of long-term exposure to heat stress and air pollution, and evaluating the interactions between adaptation and mitigation strategies. Because national boundaries do not limit most climate-related factors expected to impact human health, we propose that adaptation/mitigation policies must have a regional scope, and therefore require collaborative efforts among EMME nations. Policy suggestions include a decisive region-wide decarbonisation, the integration of environmentally driven morbidity and mortality data throughout the region, advancing the development and widespread use of affordable technologies for the production and management of drinking water by non-traditional means, the development of comprehensive strategies to improve the health status of displaced populations, and fostering regional networks for monitoring and controlling the spread of infectious diseases and disease vectors.

The circadian clock modulates Anopheles gambiae infection with Plasmodium falciparum.

Key behaviours, physiologies and gene expressions in Anopheles mosquitoes impact the transmission of Plasmodium. Such mosquito factors are rhythmic to closely follow diel rhythms. Here, we set to explore the impact of the mosquito circadian rhythm on the tripartite interaction between the vector, the parasite and the midgut microbiota, and investigate how this may affect the parasite infection outcomes. We assess Plasmodium falciparum infection prevalence and intensity, as a proxy for gametocyte infectivity, in Anopheles gambiae mosquitoes that received a gametocyte-containing bloodfeed and measure the abundance of the midgut microbiota at different times of the mosquito rearing light-dark cycle. Gametocyte infectivity is also compared in mosquitoes reared and maintained under a reversed light-dark regime. The effect of the circadian clock on the infection outcome is also investigated through silencing of the CLOCK gene that is central in the regulation of animal circadian rhythms. The results reveal that the A. gambiae circadian cycle plays a key role in the intensity of infection of P. falciparum gametocytes. We show that parasite gametocytes are more infectious during the night-time, where standard membrane feeding assays (SMFAs) at different time points in the mosquito natural circadian rhythm demonstrate that gametocytes are more infectious when ingested at midnight than midday. When mosquitoes were cultured under a reversed light/dark regime, disrupting their natural physiological homeostasis, and infected with P. falciparum at evening hours, the infection intensity and prevalence were significantly decreased. Similar results were obtained in mosquitoes reared under the standard light/dark regime upon silencing of CLOCK, a key regulator of the circadian rhythm, highlighting the importance of the circadian rhythm for the mosquito vectorial capacity. At that time, the mosquito midgut microbiota load is significantly reduced, while the expression of lysozyme C-1 (LYSC-1) is elevated, which is involved in both the immune response and microbiota digestion. We conclude that the tripartite interactions between the mosquito vector, the malaria parasite and the mosquito gut microbiota are finely tuned to support and maintain malaria transmission. Our data add to the knowledge framework required for designing appropriate and biologically relevant SMFA protocols.

Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development.

Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, magainin 2 and melittin. This small genetic modification, capable of efficient nonautonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites, while it simultaneously reduces the life span of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of transmission intensities.

Testing non-autonomous antimalarial gene drive effectors using self-eliminating drivers in the African mosquito vector Anopheles gambiae.

Gene drives for mosquito population modification are novel tools for malaria control. Strategies to safely test antimalarial effectors in the field are required. Here, we modified the Anopheles gambiae zpg locus to host a CRISPR/Cas9 integral gene drive allele (zpgD) and characterized its behaviour and resistance profile. We found that zpgD dominantly sterilizes females but can induce efficient drive at other loci when it itself encounters resistance. We combined zpgD with multiple previously characterized non-autonomous payload drives and found that, as zpgD self-eliminates, it leads to conversion of mosquito cage populations at these loci. Our results demonstrate how self-eliminating drivers could allow safe testing of non-autonomous effector-traits by local population modification. They also suggest that after engendering resistance, gene drives intended for population suppression could nevertheless serve to propagate subsequently released non-autonomous payload genes, allowing modification of vector populations initially targeted for suppression.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) represents the 2019 merger of VectorBase with the EuPathDB projects. As a Bioinformatics Resource Center funded by the National Institutes of Health, with additional support from the Welllcome Trust, VEuPathDB supports >500 organisms comprising invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Designed to empower researchers with access to Omics data and bioinformatic analyses, VEuPathDB projects integrate >1700 pre-analysed datasets (and associated metadata) with advanced search capabilities, visualizations, and analysis tools in a graphic interface. Diverse data types are analysed with standardized workflows including an in-house OrthoMCL algorithm for predicting orthology. Comparisons are easily made across datasets, data types and organisms in this unique data mining platform. A new site-wide search facilitates access for both experienced and novice users. Upgraded infrastructure and workflows support numerous updates to the web interface, tools, searches and strategies, and Galaxy workspace where users can privately analyse their own data. Forthcoming upgrades include cloud-ready application architecture, expanded support for the Galaxy workspace, tools for interrogating host-pathogen interactions, and improved interactions with affiliated databases (ClinEpiDB, MicrobiomeDB) and other scientific resources, and increased interoperability with the Bacterial & Viral BRC.

Analysis of pir gene expression across the Plasmodium life cycle.

BACKGROUND: Plasmodium interspersed repeat (pir) is the largest multigene family in the genomes of most Plasmodium species. A variety of functions for the PIR proteins which they encode have been proposed, including antigenic variation, immune evasion, sequestration and rosetting. However, direct evidence for these is lacking. The repetitive nature of the family has made it difficult to determine function experimentally. However, there has been some success in using gene expression studies to suggest roles for some members in virulence and chronic infection. METHODS: Here pir gene expression was examined across the life cycle of Plasmodium berghei using publicly available RNAseq data-sets, and at high resolution in the intraerythrocytic development cycle using new data from Plasmodium chabaudi. RESULTS: Expression of pir genes is greatest in stages of the parasite which invade and reside in red blood cells. The marked exception is that liver merozoites and male gametocytes produce a very large number of pir gene transcripts, notably compared to female gametocytes, which produce relatively few. Within the asexual blood stages different subfamilies peak at different times, suggesting further functional distinctions. Representing a subfamily of its own, the highly conserved ancestral pir gene warrants further investigation due to its potential tractability for functional investigation. It is highly transcribed in multiple life cycle stages and across most studied Plasmodium species and thus is likely to play an important role in parasite biology. CONCLUSIONS: The identification of distinct expression patterns for different pir genes and subfamilies is likely to provide a basis for the design of future experiments to uncover their function.

Unravelling population structure heterogeneity within the genome of the malaria vector Anopheles gambiae.

BACKGROUND: Whole genome re-sequencing provides powerful data for population genomic studies, allowing robust inferences of population structure, gene flow and evolutionary history. For the major malaria vector in Africa, Anopheles gambiae, other genetic aspects such as selection and adaptation are also important. In the present study, we explore population genetic variation from genome-wide sequencing of 765 An. gambiae and An. coluzzii specimens collected from across Africa. We used t-SNE, a recently popularized dimensionality reduction method, to create a 2D-map of An. gambiae and An. coluzzii genes that reflect their population structure similarities. RESULTS: The map allows intuitive navigation among genes distributed throughout the so-called "mainland" and numerous surrounding "island-like" gene clusters. These gene clusters of various sizes correspond predominantly to low recombination genomic regions such as inversions and centromeres, and also to recent selective sweeps. Because this mosquito species complex has been studied extensively, we were able to support our interpretations with previously published findings. Several novel observations and hypotheses are also made, including selective sweeps and a multi-locus selection event in Guinea-Bissau, a known intense hybridization zone between An. gambiae and An. coluzzii. CONCLUSIONS: Our results present a rich dataset that could be utilized in functional investigations aiming to shed light onto An. gambiae s.l genome evolution and eventual speciation. In addition, the methodology presented here can be used to further characterize other species not so well studied as An. gambiae, shortening the time required to progress from field sampling to the identification of genes and genomic regions under unique evolutionary processes.

Anopheles coluzzii stearoyl-CoA desaturase is essential for adult female survival and reproduction upon blood feeding.

Vitellogenesis and oocyte maturation require anautogenous female Anopheles mosquitoes to obtain a bloodmeal from a vertebrate host. The bloodmeal is rich in proteins that are readily broken down into amino acids in the midgut lumen and absorbed by the midgut epithelial cells where they are converted into lipids and then transported to other tissues including ovaries. The stearoyl-CoA desaturase (SCD) plays a pivotal role in this process by converting saturated (SFAs) to unsaturated (UFAs) fatty acids; the latter being essential for maintaining cell membrane fluidity amongst other housekeeping functions. Here, we report the functional and phenotypic characterization of SCD1 in the malaria vector mosquito Anopheles coluzzii. We show that RNA interference (RNAi) silencing of SCD1 and administration of sterculic acid (SA), a small molecule inhibitor of SCD1, significantly impact on the survival and reproduction of female mosquitoes following blood feeding. Microscopic observations reveal that the mosquito thorax is quickly filled with blood, a phenomenon likely caused by the collapse of midgut epithelial cell membranes, and that epithelial cells are depleted of lipid droplets and oocytes fail to mature. Transcriptional profiling shows that genes involved in protein, lipid and carbohydrate metabolism and immunity-related genes are the most affected by SCD1 knock down (KD) in blood-fed mosquitoes. Metabolic profiling reveals that these mosquitoes exhibit increased amounts of saturated fatty acids and TCA cycle intermediates, highlighting the biochemical framework by which the SCD1 KD phenotype manifests as a result of a detrimental metabolic syndrome. Accumulation of SFAs is also the likely cause of the potent immune response observed in the absence of infection, which resembles an auto-inflammatory condition. These data provide insights into mosquito bloodmeal metabolism and lipid homeostasis and could inform efforts to develop novel interventions against mosquito-borne diseases.

Identification of Three Novel Plasmodium Factors Involved in Ookinete to Oocyst Developmental Transition.

Plasmodium falciparum malaria remains a major cause of global morbidity and mortality, mainly in sub-Saharan Africa. The numbers of new malaria cases and deaths have been stable in the last years despite intense efforts for disease elimination, highlighting the need for new approaches to stop disease transmission. Further understanding of the parasite transmission biology could provide a framework for the development of such approaches. We phenotypically and functionally characterized three novel genes, PIMMS01, PIMMS57, and PIMMS22, using targeted disruption of their orthologs in the rodent parasite Plasmodium berghei. PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. Our results enrich the molecular understanding of the parasite-vector interactions and identify PIMMS01, PIMMS57, and PIMMS22 as new targets of transmission blocking interventions.

Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement.

Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an acceptable testing pathway of gene drives for malaria eradication.

Anopheles arabiensis hotspots along intermittent rivers drive malaria dynamics in semi-arid areas of Central Ethiopia.

BACKGROUND: Understanding malaria vector's population dynamics and their spatial distribution is important to define when and where the largest infection risks occur and implement appropriate control strategies. In this study, the seasonal spatio-temporal dynamics of the malaria vector population and transmission intensity along intermittent rivers in a semi-arid area of central Ethiopia were investigated. METHODS: Mosquitoes were collected monthly from five clusters, 2 close to a river and 3 away from a river, using pyrethrum spray catches from November 2014 to July 2016. Mosquito abundance was analysed by the mixed Poisson regression model. The human blood index and sporozoite rate was compared between seasons by a logistic regression model. RESULTS: A total of 2784 adult female Anopheles gambiae sensu lato (s.l.) were collected during the data collection period. All tested mosquitoes (n = 696) were identified as Anopheles arabiensis by polymerase chain reaction. The average daily household count was significantly higher (P = 0.037) in the clusters close to the river at 5.35 (95% CI 2.41-11.85) compared to the clusters away from the river at 0.033 (95% CI 0.02-0.05). Comparing the effect of vicinity of the river by season, a significant effect of closeness to the river was found during the dry season (P = 0.027) and transition from dry to wet season (P = 0.032). Overall, An. arabiensis had higher bovine blood index (62.8%) as compared to human blood index (23.8%), ovine blood index (9.2%) and canine blood index (0.1%). The overall sporozoite rate was 3.9% and 0% for clusters close to and away from the river, respectively. The overall Plasmodium falciparum and Plasmodium vivax entomologic inoculation rates for An. arabiensis in clusters close to the river were 0.8 and 2.2 infective bites per person/year, respectively. CONCLUSION: Mosquito abundance and malaria transmission intensity in clusters close to the river were higher which could be attributed to the riverine breeding sites. Thus, vector control interventions including targeted larval source management should be implemented to reduce the risk of malaria infection in the area.

Plasmodium oocysts respond with dormancy to crowding and nutritional stress.

Malaria parasites develop as oocysts in the mosquito for several days before they are able to infect a human host. During this time, mosquitoes take bloodmeals to replenish their nutrient and energy reserves needed for flight and reproduction. We hypothesized that these bloodmeals are critical for oocyst growth and that experimental infection protocols, typically involving a single bloodmeal at the time of infection, cause nutritional stress to the developing oocysts. Therefore, enumerating oocysts disregarding their growth and differentiation state may lead to erroneous conclusions about the efficacy of transmission blocking interventions. Here, we examine this hypothesis in Anopheles coluzzii mosquitoes infected with the human and rodent parasites Plasmodium falciparum and Plasmodium berghei, respectively. We show that oocyst growth and maturation rates decrease at late developmental stages as infection intensities increase; an effect exacerbated at very high infection intensities but fully restored with post infection bloodmeals. High infection intensities and starvation conditions reduce RNA Polymerase III activity in oocysts unless supplemental bloodmeals are provided. Our results suggest that oocysts respond to crowding and nutritional stress with a dormancy-like strategy, which urges the development of alternative methods to assess the efficacy of transmission blocking interventions.

Climate change impacts on infectious diseases in the Eastern Mediterranean and the Middle East (EMME)-risks and recommendations.

The Eastern Mediterranean and Middle East (EMME) region has rapid population growth, large differences in socio-economic levels between developed and developing countries, migration, increased water demand, and ecosystems degradation. The region is experiencing a significant warming trend with longer and warmer summers, increased frequency and severity of heat waves, and a drier climate. While climate change plays an important role in contributing to political instability in the region through displacement of people, food insecurity, and increased violence, it also increases the risks of vector-, water-, and food-borne diseases. Poorer and less educated people, young children and the elderly, migrants, and those with long-term health problems are at highest risk. A result of the inequalities among EMME countries is an inconsistency in the availability of reliable evidence about the impacts on infectious diseases. To help address this gap, a search of the literature was conducted as a basis for related recommended responses and suggested actions for preparedness and prevention. Since climate change already impacts the health of vulnerable populations in the EMME and will have a greater impact in future years, risk assessment and timely design and implementation of health preparedness and adaptation strategies are essential. Joint national and cross-border infectious diseases management systems for more effective preparedness and prevention are needed, supported by interventions that improve the environment. Without such cooperation and effective interventions, climate change will lead to an increasing morbidity and mortality in the EMME from infectious diseases, with a higher risk for the most vulnerable populations.

Antibiotic Treatment in Anopheles coluzzii Affects Carbon and Nitrogen Metabolism.

The mosquito microbiota reduces the vector competence of Anopheles to Plasmodium and affects host fitness; it is therefore considered as a potential target to reduce malaria transmission. While immune induction, secretion of antimicrobials and metabolic competition are three typical mechanisms of microbiota-mediated protection against invasive pathogens in mammals, the involvement of metabolic competition or mutualism in mosquito-microbiota and microbiota-Plasmodium interactions has not been investigated. Here, we describe a metabolome analysis of the midgut of Anopheles coluzzii provided with a sugar-meal or a non-infectious blood-meal, under conventional or antibiotic-treated conditions. We observed that the antibiotic treatment affects the tricarboxylic acid cycle and nitrogen metabolism, notably resulting in decreased abundance of free amino acids. Linking our results with published data, we identified pathways which may participate in microbiota-Plasmodium interactions via metabolic interactions or immune modulation and thus would be interesting candidates for future functional studies.