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Within-host survival and between-host transmission are key life-history traits of single-celled malaria parasites. Understanding the evolutionary forces that shape these traits is crucial to predict malaria epidemiology, drug resistance, and virulence. However, very little is known about how Plasmodium parasites adapt to their mosquito vectors. Here, we examine the evolution of the time Plasmodium parasites require to develop within the vector (extrinsic incubation period) with an individual-based model of malaria transmission that includes mosquito metabolism. Specifically, we model the metabolic cascade of resource allocation induced by blood-feeding, as well as the influence of multiple blood meals on parasite development. Our model predicts that successful vector-to-human transmission events are rare, and are caused by long-lived mosquitoes. Importantly, our results show that the life-history strategies of malaria parasites depend on the mosquito's metabolic status. In our model, additional resources provided by multiple blood meals lead to selection for parasites with slow or intermediate developmental time. These results challenge the current assumption that evolution favors fast developing parasites to maximize their chances to complete their within-mosquito life cycle. We propose that the long sporogonic cycle observed for Plasmodium is not a constraint but rather an adaptation to increase transmission potential., (© 2023. The Author(s).)

Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C-terminal domain. Two mAbs recognized linear epitopes in the C-terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the α-thrombospondin repeat (α-TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+/CS.T3 region in the α-TSR was associated with IGHV3-21/IGVL3-21 or IGLV3-1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class-switching compared to anti-repeat mAbs, live sporozoite binding and inhibitory activity was limited to a single C-linker reactive mAb with cross-reactivity to the central repeat and junction. The data provide novel insights in the human anti-C-linker and anti-α-TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The development of an effective and durable vaccine remains a central goal in the fight against malaria. Circumsporozoite protein (CSP) is the major surface protein of sporozoites and the target of the only licensed Plasmodium falciparum (Pf) malaria vaccine, RTS,S/AS01. However, vaccine efficacy is low and short-lived, highlighting the need for a second-generation vaccine with superior efficacy and durability. Here, we report a Helicobacter pylori apoferritin-based nanoparticle immunogen that elicits strong B cell responses against PfCSP epitopes that are targeted by the most potent human monoclonal antibodies. Glycan engineering of the scaffold and fusion of an exogenous T cell epitope enhanced the anti-PfCSP B cell response eliciting strong, long-lived and protective humoral immunity in mice. Our study highlights the power of rational vaccine design to generate a highly efficacious second-generation anti-infective malaria vaccine candidate and provides the basis for its further development., (© 2023. The Author(s).)

Antibodies targeting the human malaria parasite Plasmodium falciparum circumsporozoite protein (PfCSP) can prevent infection and disease. PfCSP contains multiple central repeating NANP motifs; some of the most potent anti-infective antibodies against malaria bind to these repeats. Multiple antibodies can bind the repeating epitopes concurrently by engaging into homotypic Fab-Fab interactions, which results in the ordering of the otherwise largely disordered central repeat into a spiral. Here, we characterize IGHV3-33/IGKV1-5-encoded monoclonal antibody (mAb) 850 elicited by immunization of transgenic mice with human immunoglobulin loci. mAb 850 binds repeating NANP motifs with picomolar affinity, potently inhibits Plasmodium falciparum (Pf) in vitro and, when passively administered in a mouse challenge model, reduces liver burden to a similar extent as some of the most potent anti-PfCSP mAbs yet described. Like other IGHV3-33/IGKV1-5-encoded anti-NANP antibodies, mAb 850 primarily utilizes its HCDR3 and germline-encoded aromatic residues to recognize its core NANP motif. Biophysical and cryo-electron microscopy analyses reveal that up to 19 copies of Fab 850 can bind the PfCSP repeat simultaneously, and extensive homotypic interactions are observed between densely-packed PfCSP-bound Fabs to indirectly improve affinity to the antigen. Together, our study expands on the molecular understanding of repeat-induced homotypic interactions in the B cell response against PfCSP for potently protective mAbs against Pf infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Kucharska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Malaria is a global health concern, and research efforts are ongoing to develop a superior vaccine to RTS,S/AS01. To guide immunogen design, we seek a comprehensive understanding of the protective humoral response against Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). In contrast to the well-studied responses to the repeat region and the C-terminus, the antibody response against the N-terminal domain of PfCSP (N-CSP) remains obscure. Here, we characterized the molecular recognition and functional efficacy of the N-CSP-specific monoclonal antibody 5D5. The crystal structure at 1.85-Å resolution revealed that 5D5 binds an α-helical epitope in N-CSP with high affinity through extensive shape and charge complementarity and the unusual utilization of an antibody N-linked glycan. Nevertheless, functional studies indicated low 5D5 binding to live Pf sporozoites and lack of sporozoite inhibition in vitro and in vivo. Overall, our data do not support the inclusion of the 5D5 N-CSP epitope into the next generation of CSP-based vaccines., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Thai et al.)

Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and propose that the mosquito complement-like system contributes to the species-specific dynamics of Plasmodium invasion., Competing Interests: The authors have declared that no competing interests exist.

The circumsporozoite protein of the human malaria parasite Plasmodium falciparum (PfCSP) is the main target of antibodies that prevent the infection and disease, as shown in animal models. However, the limited efficacy of the PfCSP-based vaccine RTS,S calls for a better understanding of the mechanisms driving the development of the most potent human PfCSP antibodies and identification of their target epitopes. By characterizing 200 human monoclonal PfCSP antibodies induced by sporozoite immunization, we establish that the most potent antibodies bind around a conserved (N/D)PNANPN(V/A) core. High antibody affinity to the core correlates with protection from parasitemia in mice and evolves around the recognition of NANP motifs. The data suggest that the rational design of a next-generation PfCSP vaccine that elicits high-affinity antibody responses against the core epitope will promote the induction of protective humoral immune responses.

The egress and fertilization of Plasmodium gametes and development of a motile ookinete are the first crucial steps that mediate the successful transmission of the malaria parasites from humans to the Anopheles vector. However, limited information exists about the cell biology and regulation of this process. Technical impediments in the establishment of in vitro conditions for ookinete maturation in Plasmodium falciparum and other human malaria parasites further constrain a detailed characterization of ookinete maturation. Here, using fluorescence microscopy and immunolabeling, we compared P. falciparum ookinete maturation in Anopheles coluzzii mosquitoes in vivo and in cell culture in vitro . Our results identified two critical steps in ookinete maturation that are regulated by distinct mosquito factors, thereby highlighting the role of the mosquito environment in the transmission efficiency of malaria parasites., (Copyright © 2020 Siciliano, Costa, Suárez-Cortés, Valleriani, Alano and Levashina.)

The blood-feeding behavior of Anopheles females delivers essential nutrients for egg development and drives parasite transmission between humans. Plasmodium growth is adapted to the vector reproductive cycle, but how changes in the reproductive cycle impact parasite development remains unclear. Here, we show that the bloodmeal-induced miR-276-5p fine-tunes the expression of branched-chain amino acid transferase to terminate the reproductive cycle. Silencing of miR-276 prolongs high rates of amino acid (AA) catabolism and increases female fertility, suggesting that timely termination of AA catabolism restricts mosquito investment into reproduction. Prolongation of AA catabolism in P. falciparum-infected females also compromises the development of the transmissible sporozoite forms. Our results suggest that Plasmodium sporogony exploits the surplus mosquito resources available after reproductive investment and demonstrate the crucial role of the mosquito AA metabolism in within-vector parasite proliferation and malaria transmission.

Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites., (Copyright © 2019 Tibúrcio et al.)

The complement-like protein thioester-containing protein 1 (TEP1) is a key factor in the immune response of the malaria vector Anopheles gambiae to pathogens. Multiple allelic variants of TEP1 have been identified in laboratory strains and in the field, and are correlated with distinct immunophenotypes. TEP1 is tightly regulated by conformational changes induced by cleavage in a protease-sensitive region. Cleaved TEP1 forms exhibit significant variation in stability from hours to days at room temperature. In particular, the refractory allele TEP1*R1 is significantly more stable than the susceptible allele TEP1*S1. This raises the question of whether the stability of cleaved TEP1 is linked to allelic variation and varying immunophenotypes. We have analyzed the stability of the cleaved form of additional TEP1 alleles and constructs. We show that stability is correlated with allelic variation within two specific loops in direct proximity to the thioester bond. The variable loops are part of an interface between the TED and MG8 domains of TEP1 that protect the thioester from hydrolysis. Engineering specific disulfide bonds to prevent separation of the TED-MG8 interface stabilizes the cleaved form of TEP1 for months at room temperature. Cleaved TEP1 forms a soluble complex with a heterodimer of two leucine-rich repeat proteins, LRIM1 and APL1C, and precipitates in the absence of this complex. The molecular structure and oligomeric state of the TEP1/LRIM1/APL1C complex is unclear. The C-terminal coiled-coil domain of the LRIM1/APL1C complex is sufficient to stabilize the cleaved form of TEP1 in solution but cleaved forms of disulfide-stabilized TEP1 do not interact with LRIM1/APL1C. This implies that formation of the TEP1cut/LRIM1/APL1C complex is related to the conformational change that induces the precipitation of cleaved TEP1., Competing Interests: MW declares the following potential competing interest: Shift Health (paid employment). AC declares the following potential competing interest: Pierre Fabre (paid employment) This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Malaria, a major cause of child mortality in Africa, is engendered by Plasmodium parasites that are transmitted by anopheline mosquitoes. Fitness of Plasmodium parasites is closely linked to the ecology and evolution of its anopheline vector. However, whether the genetic structure of vector populations impacts malaria transmission remains unknown. Here, we describe a partitioning of the African malaria vectors into generalists and specialists that evolve along ecological boundaries. We next identify the contribution of mosquito species to Plasmodium abundance using Granger causality tests for time-series data collected over two rainy seasons in Mali. We find that mosquito microevolution, defined by changes in the genetic structure of a population over short ecological timescales, drives Plasmodium dynamics in nature, whereas vector abundance, infection prevalence, temperature and rain have low predictive values. Our study demonstrates the power of time-series approaches in vector biology and highlights the importance of focusing local vector control strategies on mosquito species that drive malaria dynamics.

Recent scientific breakthroughs have significantly expanded our understanding of arthropod vector immunity. Insights in the laboratory have demonstrated how the immune system provides resistance to infection, and in what manner innate defenses protect against a microbial assault. Less understood, however, is the effect of biotic and abiotic factors on microbial-vector interactions and the impact of the immune system on arthropod populations in nature. Furthermore, the influence of genetic plasticity on the immune response against vector-borne pathogens remains mostly elusive. Herein, we discuss evolutionary forces that shape arthropod vector immunity. We focus on resistance, pathogenicity and tolerance to infection. We posit that novel scientific paradigms should emerge when molecular immunologists and evolutionary ecologists work together., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Malaria is a fatal human parasitic disease transmitted by a mosquito vector. Although the evolution of within-host malaria virulence has been the focus of many theoretical and empirical studies, the vector's contribution to this process is not well understood. Here, we explore how within-vector resource exploitation would impact the evolution of within-host Plasmodium virulence. By combining within-vector dynamics and malaria epidemiology, we develop a mathematical model, which predicts that non-competitive parasitic resource exploitation within-vector restricts within-host parasite virulence. To validate our model, we experimentally manipulate mosquito lipid trafficking and gauge within-vector parasite development and within-host infectivity and virulence. We find that mosquito-derived lipids determine within-host parasite virulence by shaping development (quantity) and metabolic activity (quality) of transmissible sporozoites. Our findings uncover the potential impact of within-vector environment and vector control strategies on the evolution of malaria virulence.

Insect immunity to extracellular microbes relies largely on the TOLL and IMD pathways. In this issue of Immunity, Goto et al. (2018) report that the IKKβ-Relish module of the IMD pathway hitches up the intracellular sensor STING to activate antiviral responses in Drosophila., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mosquito blood cells are immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond morphological and functional criteria used for their classification. Here, we combined the power of single-cell RNA sequencing, high-content imaging flow cytometry, and single-molecule RNA hybridization to analyze a subset of blood cells of the malaria mosquito Anopheles gambiae By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional program. Analyses of differentially expressed genes identified transcriptional signatures of two cell types and provide insights into the current classification of these cells. We further demonstrate the active transfer of a cellular marker between blood cells that may confound their identification. We propose that cell-to-cell exchange may contribute to cellular diversity and functional plasticity seen across biological systems., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens. We determined the molecular mechanism underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite Plasmodium falciparum (PfCSP). We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Affinity maturation, the clonal selection and expansion of antigen-activated B cells expressing somatically mutated antibody variants that develop during T cell-dependent germinal center reactions, is considered pivotal for efficient development of protective B cell memory responses to infection and vaccination. Repeated antigen exposure promotes affinity maturation but each time also recruits antigen-reactive naïve B cells into the response. Here, we determined the relative impact of affinity maturation versus antigen-mediated clonal selection of naïve B cells to mount potent B cell memory responses in humans after repeated exposure to a complex pathogen, the malaria parasite Plasmodium falciparum (Pf). Using single-cell immunoglobulin (Ig) gene sequencing and production of recombinant monoclonal antibodies, we analyzed the origin, development, and quality of memory B cell responses to Pf circumsporozoite protein (PfCSP), the major sporozoite surface protein. We show that after repeated immunization of Pf-naïve volunteers with infectious Pf sporozoites (PfSPZ Challenge) under chloroquine prophylaxis (PfSPZ-CVac), the clonal selection of potent germline and memory B cell precursors against the central PfCSP NANP repeat outpaces affinity maturation because the majority of Ig gene mutations are affinity-neutral. Mathematical modeling explains how the efficiency of affinity maturation decreases strongly with antigen complexity. Thus, in the absence of long-term exposure, the frequency of antigen-reactive precursors and likelihood of their activation rather than affinity maturation will determine the quality of anti-PfCSP memory B cell responses. These findings have wide implications for the design of vaccination strategies to induce potent B cell memory responses against PfCSP and presumably other structurally complex antigens., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Anopheles gambiae mosquitoes transmit the human malaria parasite Plasmodium falciparum , which causes the majority of fatal malaria cases worldwide. The hematophagous lifestyle defines mosquito reproductive biology and is exploited by P. falciparum for its own sexual reproduction and transmission. The two main phases of the mosquito reproductive cycle, previtellogenic (PV) and postblood meal (PBM), shape its capacity to transmit malaria. Transition between these phases is tightly coordinated to ensure homeostasis between mosquito tissues and successful reproduction. One layer of control is provided by microRNAs (miRNAs), well-known regulators of blood meal digestion and egg development in Aedes mosquitoes. Here, we report a global overview of tissue-specific miRNAs (miRNA) expression during the PV and PBM phases and identify miRNAs regulated during PV to PBM transition. The observed coordinated changes in the expression levels of a set of miRNAs in the energy-storing tissues suggest a role in the regulation of blood meal-induced metabolic changes., (Copyright © 2018 Lampe, Levashina.)

Antibodies against the central repeat of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) inhibit parasite activity and correlate with protection from malaria. However, the humoral response to the PfCSP C terminus (C-PfCSP) is less well characterized. Here, we describe B cell responses to C-PfCSP from European donors who underwent immunization with live Pf sporozoites (PfSPZ Challenge) under chloroquine prophylaxis (PfSPZ-CVac), and were protected against controlled human malaria infection. Out of 215 PfCSP-reactive monoclonal antibodies, only two unique antibodies were specific for C-PfCSP, highlighting the rare occurrence of C-PfCSP-reactive B cells in PfSPZ-CVac-induced protective immunity. These two antibodies showed poor sporozoite binding and weak inhibition of parasite traversal and development, and did not protect mice from infection with PfCSP transgenic Plasmodium berghei sporozoites. Structural analyses demonstrated that one antibody interacts with a polymorphic region overlapping two T cell epitopes, suggesting that variability in C-PfCSP may benefit parasite escape from humoral and cellular immunity. Our data identify important features underlying C-PfCSP shortcomings as a vaccine target., (© 2018 Scally et al.)

Antibodies against the NANP repeat of circumsporozoite protein (CSP), the major surface antigen of Plasmodium falciparum (Pf) sporozoites, can protect from malaria in animal models but protective humoral immunity is difficult to induce in humans. Here we cloned and characterized rare affinity-matured human NANP-reactive memory B cell antibodies elicited by natural Pf exposure that potently inhibited parasite transmission and development in vivo. We unveiled the molecular details of antibody binding to two distinct protective epitopes within the NANP repeat. NANP repeat recognition was largely mediated by germline encoded and immunoglobulin (Ig) heavy-chain complementarity determining region 3 (HCDR3) residues, whereas affinity maturation contributed predominantly to stabilizing the antigen-binding site conformation. Combined, our findings illustrate the power of exploring human anti-CSP antibody responses to develop tools for malaria control in the mammalian and the mosquito vector and provide a molecular basis for the structure-based design of next-generation CSP malaria vaccines., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The blood feeding requirements of insects are often exploited by pathogens for their transmission. This is also the case of the protozoan parasites of genus Plasmodium , the causative agents of malaria. Every year malaria claims the lives of a half million people, making its vector, the Anopheles mosquito, the deadliest animal in the world. However, mosquitoes mount powerful immune responses that efficiently limit parasite proliferation. Among the immune signaling pathways identified in the main malaria vector Anopheles gambiae , the NF-κB-like signaling cascades REL2 and REL1 are essential for eliciting proper immune reactions, but only REL2 has been implicated in the responses against the human malaria parasite Plasmodium falciparum . Instead, constitutive activation of REL1 causes massive killing of rodent malaria parasites. In this review, we summarize our present knowledge on the REL2 pathway in Anopheles mosquitoes and its role in mosquito immune responses to diverse pathogens, with a focus on Plasmodium . Mosquito-parasite interactions are crucial for malaria transmission and, therefore, represent a potential target for malaria control strategies.

In the last years, microRNAs (miRNAs) have been established as important post-transcriptional regulators of critical physiological processes in animals and plants. Here, we summarize what is known about miRNA biosynthesis, expression and function in the malaria vector mosquito Anopheles gambiae with a particular emphasis on the mosquito-parasite interactions. We discuss the important gaps in the current knowledge, including the potential of miRNA manipulation for future vector control strategies., (© 2017 John Wiley & Sons Ltd.)

An essential step in the transmission of the malaria parasite to the Anopheles vector is the transformation of the mature gametocytes into gametes in the mosquito gut, where they egress from the erythrocytes and mate to produce a zygote, which matures into a motile ookinete. Osmiophilic bodies are electron dense secretory organelles of the female gametocytes which discharge their contents during gamete formation, suggestive of a role in gamete egress. Only one protein with no functional annotation, Pfg377, is described to specifically reside in osmiophilic bodies in Plasmodium falciparum Importantly, Pfg377 defective gametocytes lack osmiophilic bodies and fail to infect mosquitoes, as confirmed here with newly produced pfg377 disrupted parasites. The unique feature of Pfg377 defective gametocytes of lacking osmiophilic bodies was here exploited to perform comparative, label free, global and affinity proteomics analyses of mutant and wild type gametocytes to identify components of these organelles. Subcellular localization studies with fluorescent reporter gene fusions and specific antibodies revealed an osmiophilic body localization for four out of five candidate gene products analyzed: the proteases PfSUB2 (subtilisin 2) and PfDPAP2 (Dipeptidyl aminopeptidase 2), the ortholog of the osmiophilic body component of the rodent malaria gametocytes PbGEST and a previously nonannotated 13 kDa protein. These results establish that osmiophilic bodies and their components are dispensable or marginally contribute (PfDPAP2) to gamete egress. Instead, this work reveals a previously unsuspected role of these organelles in P. falciparum development in the mosquito vector., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Vector-borne pathogens impact public health, animal production, and animal welfare. Research on arthropod vectors such as mosquitoes, ticks, sandflies, and midges which transmit pathogens to humans and economically important animals is crucial for development of new control measures that target transmission by the vector. While insecticides are an important part of this arsenal, appearance of resistance mechanisms is increasingly common. Novel tools for genetic manipulation of vectors, use of Wolbachia endosymbiotic bacteria, and other biological control mechanisms to prevent pathogen transmission have led to promising new intervention strategies, adding to strong interest in vector biology and genetics as well as vector-pathogen interactions. Vector research is therefore at a crucial juncture, and strategic decisions on future research directions and research infrastructure investment should be informed by the research community. A survey initiated by the European Horizon 2020 INFRAVEC-2 consortium set out to canvass priorities in the vector biology research community and to determine key activities that are needed for researchers to efficiently study vectors, vector-pathogen interactions, as well as access the structures and services that allow such activities to be carried out. We summarize the most important findings of the survey which in particular reflect the priorities of researchers in European countries, and which will be of use to stakeholders that include researchers, government, and research organizations.

Microbes play key roles in shaping the physiology of insects and can influence behavior, reproduction and susceptibility to pathogens. In Sub-Saharan Africa, two major malaria vectors, Anopheles gambiae and An. coluzzii, breed in distinct larval habitats characterized by different microorganisms that might affect their adult physiology and possibly Plasmodium transmission. We analyzed the reproductive microbiomes of male and female An. gambiae and An. coluzzii couples collected from natural mating swarms in Burkina Faso. 16S rRNA sequencing on dissected tissues revealed that the reproductive tracts harbor a complex microbiome characterized by a large core group of bacteria shared by both species and all reproductive tissues. Interestingly, we detected a significant enrichment of several gender-associated microbial biomarkers in specific tissues, and surprisingly, similar classes of bacteria in males captured from one mating swarm, suggesting that these males originated from the same larval breeding site. Finally, we identified several endosymbiotic bacteria, including Spiroplasma, which have the ability to manipulate insect reproductive success. Our study provides a comprehensive analysis of the reproductive microbiome of important human disease vectors, and identifies a panel of core and endosymbiotic bacteria that can be potentially exploited to interfere with the transmission of malaria parasites by the Anopheles mosquito.

Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3-5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites.

Thioester-containing protein 1 (TEP1) is a key immune factor that determines mosquito resistance to a wide range of pathogens, including malaria parasites. Here we report a new allele-specific function of TEP1 in male fertility. We demonstrate that during spermatogenesis TEP1 binds to and removes damaged cells through the same complement-like cascade that kills malaria parasites in the mosquito midgut. Further, higher fertility rates are mediated by an allele that renders the mosquito susceptible to Plasmodium. By elucidating the molecular and genetic mechanisms underlying TEP1 function in spermatogenesis, our study suggests that pleiotropic antagonism between reproduction and immunity may shape resistance of mosquito populations to malaria parasites.

Transgenesis is an essential tool to investigate gene function and to introduce desired characters in laboratory organisms. Setting-up transgenesis in non-model organisms is challenging due to the diversity of biological life traits and due to knowledge gaps in genomic information. Some procedures will be broadly applicable to many organisms, and others have to be specifically developed for the target species. Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects. Here, we report a compilation of the transgenesis tools that we have designed for the malaria vector Anopheles gambiae, including new docking strains, convenient transgenesis plasmids, a puromycin resistance selection marker, mosquitoes expressing cre recombinase, and various reporter lines defining the activity of cloned promoters. This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis. Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects., (Copyright © 2015 Volohonsky et al.)

Malaria, the human infectious disease caused by Plasmodium parasites, is transmitted by the bite of the mosquito Anopheles gambiae. Mosquitoes actively detect Plasmodium and mount efficient responses that eliminate the majority of invading parasites. Such responses include hemocyte-mediated defenses, activation of the complement-like system, melanization, and immune signaling cascades. This review aims to summarize our current knowledge of the mosquito immune responses to Plasmodium and to highlight the remaining gaps in our understanding of these events., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Wolbachia are maternally transmitted intracellular bacteria that invade insect populations by manipulating their reproduction and immunity and thus limiting the spread of numerous human pathogens. Experimental Wolbachia infections can reduce Plasmodium numbers in Anopheles mosquitoes in the laboratory, however, natural Wolbachia infections in field anophelines have never been reported. Here we show evidence of Wolbachia infections in Anopheles gambiae in Burkina Faso, West Africa. Sequencing of the 16S rRNA gene identified Wolbachia sequences in both female and male germlines across two seasons, and determined that these sequences are vertically transmitted from mother to offspring. Whole-genome sequencing of positive samples suggests that the genetic material identified in An. gambiae belongs to a novel Wolbachia strain, related to but distinct from strains infecting other arthropods. The evidence of Wolbachia infections in natural Anopheles populations promotes further investigations on the possible use of natural Wolbachia-Anopheles associations to limit malaria transmission.

Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.

Anopheline mosquitoes are the only vectors of human malaria worldwide. It is now widely accepted that mosquito immune responses play a crucial role in restricting Plasmodium development within the vector; therefore, further dissection of the molecular mechanisms underlying these processes should inform new vector control strategies urgently needed to roll back the disease. Here, using genome-wide transcriptional profiling, bioinformatics, and functional gene analysis, we identify a new axis of mosquito resistance to monoclonal Plasmodium falciparum infections that includes the AP-1 transcription factor Fos and the transglutaminase 2 (TGase2), a cross-linking enzyme with known roles in wound responses. We demonstrate that Fos regulates induction of TGase2 expression after wounding but does not affect expression of the components of the well characterized complement-like system. Silencing of Fos or of TGase2 aborts the wounding-induced mosquito killing of P. falciparum. These results reveal multiple signaling pathways that are required for efficient Plasmodium killing in Anopheles gambiae.

Anopheles gambiae mosquitoes are the major vectors of human malaria parasites. However, mosquitoes are not passive hosts for parasites, actively limiting their development in vivo. Our current understanding of the mosquito antiparasitic response is mostly based on the phenotypic analysis of gene knockdowns obtained by RNA interference (RNAi), through the injection or transfection of long dsRNAs in adult mosquitoes or cultured cells, respectively. Recently, RNAi has been extended to silence specifically one allele of a given gene in a heterozygous context, thus allowing to compare the contribution of different alleles to a phenotype in the same genetic background.

Background: Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures., Methods: A method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females., Results: Fluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers., Conclusions: The developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors.

Mosquito infections with natural isolates of Plasmodium falciparum are notoriously variable and pose a problem for reliable evaluation of efficiency of transmission-blocking agents for malaria control interventions. Here, we show that monoclonal P. falciparum isolates produce higher parasite loads than mixed ones. Induction of the mosquito immune responses by wounding efficiently decreases Plasmodium numbers in monoclonal infections but fails to do so in infections with two or more parasite genotypes. Our results point to the parasites genetic complexity as a potentially crucial component of mosquito-parasite interactions., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines.

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.

Vector-borne diseases, transmitted by bloodsucking arthropods, pose worldwide socio-medical and economical problems. Some of the major human infectious diseases, such as malaria, Dengue fever, and yellow fever, are transmitted by mosquitoes. While the majority of pathogens enjoy extracellular life styles in insects, viruses and some endosymbionts are strictly intracellular. Here, we summarize our knowledge on defense reactions against intracellular microorganisms in dipteran insects and discuss the potential of insects as models to study human pathogens., (© 2011 John Wiley & Sons A/S.)

Ticks are important ectoparasites and vectors of multiple human and animal diseases. The obligatory hemophagy of ticks provides a formidable route for parasite transmission from one host to another. Parasite survival inside the tick relies on the ability of a pathogen to escape or inhibit tick immune defenses, but the molecular interactions between the tick and its pathogens remain poorly understood. Here we report that tick genomes are unique in that they contain all known classes of the α(2)-macroglobulin family (α(2)M-F) proteins: α(2)-macroglobulin pan-protease inhibitors, C3 complement components, and insect thioester-containing and macroglobulin-related proteins. By using RNA interference-mediated gene silencing in the hard tick Ixodes ricinus we demonstrated the central role of a C3-like molecule in the phagocytosis of bacteria and revealed nonredundant functions for α(2)M-F proteins. Assessment of α(2)M-F functions in a single organism should significantly contribute to the general knowledge on the evolution and function of the complement system. Importantly, understanding the tick immune mechanisms should provide new concepts for efficient transmission blocking of tick-borne diseases., (Copyright © 2011 S. Karger AG, Basel.)

The leucine-rich repeat (LRR) proteins LRIM1 and APL1C control the function of the complement-like protein TEP1 in Anopheles mosquitoes. The molecular structure of LRIM1 and APL1C and the basis of their interaction with TEP1 represent a new type of innate immune complex. The LRIM1/APL1C complex specifically binds and solubilizes a cleaved form of TEP1 without an intact thioester bond. The LRIM1 and APL1C LRR domains have a large radius of curvature, glycosylated concave face, and a novel C-terminal capping motif. The LRIM1/APL1C complex is a heterodimer with a single intermolecular disulfide bond. The structure of the LRIM1/APL1C heterodimer reveals an interface between the two LRR domains and an extensive C-terminal coiled-coil domain. We propose that a cleaved form of TEP1 may act as a convertase for activation of other TEP1 molecules and that the LRIM1/APL1C heterodimer regulates formation of this TEP1 convertase.

When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes., Competing Interests: The authors have declared that no competing interests exist.

Malaria is an infectious disease caused by Plasmodium and transmitted to humans by the Anopheles mosquitoes. The mosquito immune system predominantly targets Plasmodium at the ookinete stage, and efficiently eliminates the majority of invading parasites. Identification of the components of the mosquito complement system now provides new focus for studies on the activation and control of this pathway, whose manipulation is expected to block malaria transmission at the vector level., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The ability of Anopheles gambiae mosquitoes to transmit Plasmodium parasites is highly variable between individuals. However, the genetic basis of this variability has remained unknown. We combined genome-wide mapping and reciprocal allele-specific RNA interference (rasRNAi) to identify the genomic locus that confers resistance to malaria parasites and demonstrated that polymorphisms in a single gene encoding the antiparasitic thioester-containing protein 1 (TEP1) explain a substantial part of the variability in parasite killing. The link between TEP1 alleles and resistance to malaria may offer new tools for controlling malaria transmission. The successful application of rasRNAi in Anopheles suggests that it could also be applied to other organisms where RNAi is feasible to dissect complex phenotypes to the level of individual quantitative trait alleles.

Plasmodium development within Anopheles mosquitoes is a vulnerable step in the parasite transmission cycle, and targeting this step represents a promising strategy for malaria control. The thioester-containing complement-like protein TEP1 and two leucine-rich repeat (LRR) proteins, LRIM1 and APL1, have been identified as major mosquito factors that regulate parasite loads. Here, we show that LRIM1 and APL1 are required for binding of TEP1 to parasites. RNAi silencing of the LRR-encoding genes results in deposition of TEP1 on Anopheles tissues, thereby depleting TEP1 from circulation in the hemolymph and impeding its binding to Plasmodium. LRIM1 and APL1 not only stabilize circulating TEP1, they also stabilize each other prior to their interaction with TEP1. Our results indicate that three major antiparasitic factors in mosquitoes jointly function as a complement-like system in parasite killing, and they reveal a role for LRR proteins as complement control factors.

Malaria is a disease that kills more than a million people each year in tropical and subtropical countries. The disease is caused by Plasmodium parasites and is transmitted to humans exclusively by mosquitoes of the genus Anopheles. The lack of functional approaches has hampered study of the biological networks that determine parasite transmission by the insect vector. The recent discovery of RNA interference and its adaptation to mosquitoes is now providing crucial tools for the dissection of vector-parasite interactions and for the analysis of aspects of mosquito biology influencing the vectorial capacity. Two RNAi approaches have been established in mosquitoes: transient gene silencing by direct injection of double-stranded RNA, and stable expression of hairpin RNAs from transgenes integrated in the genome. Here we describe these methods in detail, providing information about their use and limitations.

Anopheles gambiae mosquitoes are the principal vectors of malaria. A major determinant of the capacity of these mosquitoes as disease vectors is their high reproductive rate. Reproduction depends on a single insemination, which profoundly changes the behavior and physiology of females. To identify factors and mechanisms relevant to the fertility of A. gambiae, we performed a comprehensive analysis of the molecular and cellular machinery associated with copulation in females. Initial whole-body microarray experiments comparing virgins with females at 2 h, 6 h, and 24 h after mating detected large transcriptional changes. Analysis of tissue localization identified a subset of genes whose expression was strikingly regulated by mating in the lower reproductive tract and, surprisingly, the gut. In the atrium of virgin females, where the male seminal fluid is received, our studies revealed a "mating machinery" consisting of molecular and structural components that are turned off or collapse after copulation, suggesting that this tissue loses its competence for further insemination. In the sperm storage organ, we detected a number of mating-responsive genes likely to have a role in the maintenance and function of stored sperm. These results identify genes and mechanisms regulating the reproductive biology of A. gambiae females, highlighting considerable differences with Drosophila melanogaster. Our data inform vector control strategies and reveal promising targets for the manipulation of fertility in field populations of these important disease vectors.