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Manipulation of the mosquito gut microbiota can lay the foundations for novel methods for disease transmission control. Mosquito blood feeding triggers a significant, transient increase of the gut microbiota, but little is known about the mechanisms by which the mosquito controls this bacterial growth whilst limiting inflammation of the gut epithelium. Here, we investigate the gut epithelial response to the changing microbiota load upon blood feeding in the malaria vector Anopheles coluzzii. We show that the synthesis and integrity of the peritrophic matrix, which physically separates the gut epithelium from its luminal contents, is microbiota dependent. We reveal that the peritrophic matrix limits the growth and persistence of Enterobacteriaceae within the gut, whilst preventing seeding of a systemic infection. Our results demonstrate that the peritrophic matrix is a key regulator of mosquito gut homeostasis and establish functional analogies between this and the mucus layers of the mammalian gastrointestinal tract., Author summary When a female mosquito takes a blood meal from a human, the bacteria residing within its gut grow significantly. Following a blood meal, female mosquitoes produce a barrier within their gut, known as the peritrophic matrix, which physically separates the blood meal from the cells of the epithelium. Here, we show that the presence of bacteria in the gut is required for the synthesis of the peritrophic matrix. By experimentally disrupting this barrier, we find that this structure plays a role in limiting the extent to which bacteria of one particular family are able to grow and persist in the mosquito gut. We also find that the peritrophic matrix ensures that bacteria remain within the gut, preventing them from invading the mosquito body cavity. These results will be useful in designing disease control strategies that depend on the ability of bacteria to colonize and persist in relevant tissues in the mosquito host.

Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component., Author Summary In malaria vector mosquitoes, the presence of bacteria and malaria parasites is tightly linked. Bacteria that are part of the mosquito gut ecosystem are critical modulators of the immune response elicited during infection with malaria parasites. Furthermore, responses against oral bacterial infections can affect malaria parasites. Here, we combined mosquito gut infections with the enterobacterium Serratia marcescens with genome-wide discovery and phenotypic analysis of genes involved in antibacterial responses to characterize molecular processes that control gut bacterial infections thus possibly affecting the mosquito susceptibility to infection by malaria parasites. Our data reveal complex genetic networks controlling the gut bacterial infection load and ecosystem homeostasis. These networks appear to exhibit much higher specificity toward specific classes of bacteria than previously thought and include behavioral response circuits involved in antibacterial immunity.

Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition., Author Summary The malaria-transmitting mosquito, Anopheles gambiae, uses a complement-like pathway to defend against Plasmodium parasites. The complement C3-like protein, TEP1, binds to the surface of invading parasites, triggering their destruction and clearance. LRIM1 and APL1C, two leucine-rich repeat proteins, form a disulfide-bonded complex which stabilizes mature TEP1 and promotes its binding to parasites. Here, we investigate the structural and biochemical features of the LRIM1/APL1C complex and its interaction with TEP1. We identify key amino acid residues responsible for covalently linking LRIM1 and APL1C and the region of the complex where TEP1 binds. Importantly, we demonstrate that the LRIM1/APL1C complex can interact with the mature form of three other TEPs, including TEP3, which we characterize as a novel Plasmodium antagonist. Our results suggest that the LRIM1/APL1C complex has a modular architecture in which distinct functions map to different regions. Our study provides important insights into how the A. gambiae complement pathway helps mosquitoes fight against the malaria parasite.

Malaria parasites must undergo sexual and sporogonic development in mosquitoes before they can infect their vertebrate hosts. We report the discovery and characterization of MISFIT, the first protein with paternal effect on the development of the rodent malaria parasite Plasmodium berghei in Anopheles mosquitoes. MISFIT is expressed in male gametocytes and localizes to the nuclei of male gametocytes, zygotes and ookinetes. Gene disruption results in mutant ookinetes with reduced genome content, microneme defects and altered transcriptional profiles of putative cell cycle regulators, which yet successfully invade the mosquito midgut. However, developmental arrest ensues during the ookinete transformation to oocysts leading to malaria transmission blockade. Genetic crosses between misfit mutant parasites and parasites that are either male or female gamete deficient reveal a strict requirement for a male misfit allele. MISFIT belongs to the family of formin-like proteins, which are known regulators of the dynamic remodeling of actin and microtubule networks. Our data identify the ookinete-to-oocyst transition as a critical cell cycle checkpoint in Plasmodium development and lead us to hypothesize that MISFIT may be a regulator of cell cycle progression. This study offers a new perspective for understanding the male contribution to malaria parasite development in the mosquito vector., Author Summary The unicellular protozoan parasites that cause malaria must undergo sexual development and subsequent proliferation in mosquitoes before they can infect humans and cause malaria. We characterized the first protein with paternal effect on the development of malaria parasites in the mosquito. This protein, which we named MISFIT, is produced in the progenitor cells of male gametes and found in the nuclei of these cells as well as in the nuclei of zygotes and their invasive forms, termed ookinetes. Disruption of the gene that encodes MISFIT leads to ookinetes with reduced DNA content, a defective secretory machinery and altered expression of various regulators of DNA replication and cell division. These mutant parasites stop developing immediately after traversing the mosquito gut, leading to malaria transmission blockage. Our study offers a new perspective for understanding the sexual development of malaria parasites in the mosquito vector, which leads to transmission of one of the most devastating diseases of mankind.

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1cut that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals., Author Summary Mosquitoes are vectors of numerous human diseases including malaria. Disease transmission requires that microbes overcome the robust mosquito immune system. In the African malaria mosquito, the TEP1 protein that is homologous to mammalian complement factor C3 is shown to play a central role in mosquito immunity to malaria parasites and bacteria. In this study, we report that another mosquito protein belonging to a class of non-catalytic enzymes that are specific to arthropods is a core component of the mosquito complement-like immune pathway. We found that this new protein, named SPCLIP1, regulates the accumulation of TEP1 on malaria parasites and bacteria, and show that this can lead to distinct defense reactions including lysis and melanization of the pathogen. This work is valuable because it reveals novel insight into the regulation of mosquito complement on microbial surfaces such as those of the malaria parasites. Unraveling the molecular mechanisms regulating these defense responses may ultimately lead to the design of novel disease blocking strategies in the vector.