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

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

The voltage-gated sodium channel, para, limits Anopheles coluzzii vector competence in a microbiota dependent manner.

The voltage-gated sodium channel, para, is a target of DDT and pyrethroid class insecticides. Single nucleotide mutations in para, called knockdown resistant or kdr, which contribute to resistance against DDT and pyrethroid insecticides, have been correlated with increased susceptibility of Anopheles to the human malaria parasite Plasmodium falciparum. However, a direct role of para activity on Plasmodium infection has not yet been established. Here, using RNA-mediated silencing, we provide in vivo direct evidence for the requirement of wild-type (wt) para function for insecticide activity of deltamethrin. Depletion of wt para, which is susceptible to insecticide, causes deltamethrin tolerance, indicating that insecticide-resistant kdr alleles are likely phenocopies of loss of para function. We then show that normal para activity in An. coluzzii limits Plasmodium infection prevalence for both P. falciparum and P. berghei. A transcriptomic analysis revealed that para activity does not modulate the expression of immune genes. However, loss of para function led to enteric dysbiosis with a significant increase in the total bacterial abundance, and we show that para function limiting Plasmodium infection is microbiota dependent. In the context of the bidirectional "enteric microbiota-brain" axis studied in mammals, these results pave the way for studying whether the activity of the nervous system could control Anopheles vector competence.

Genetics and immunity of Anopheles response to the entomopathogenic fungus Metarhizium anisopliae overlap with immunity to Plasmodium.

Entomopathogenic fungi have been explored as a potential biopesticide to counteract the insecticide resistance issue in mosquitoes. However, little is known about the possibility that genetic resistance to fungal biopesticides could evolve in mosquito populations. Here, we detected an important genetic component underlying Anopheles coluzzii survival after exposure to the entomopathogenic fungus Metarhizium anisopliae. A familiality study detected variation for survival among wild mosquito isofemale pedigrees, and genetic mapping identified two loci that significantly influence mosquito survival after fungus exposure. One locus overlaps with a previously reported locus for Anopheles susceptibility to the human malaria parasite Plasmodium falciparum. Candidate gene studies revealed that two LRR proteins encoded by APL1C and LRIM1 genes in this newly mapped locus are required for protection of female A. coluzzii from M. anisopliae, as is the complement-like factor Tep1. These results indicate that natural Anopheles populations already segregate frequent genetic variation for differential mosquito survival after fungal challenge and suggest a similarity in Anopheles protective responses against fungus and Plasmodium. However, this immune similarity raises the possibility that fungus-resistant mosquitoes could also display enhanced resistance to Plasmodium, suggesting an advantage of selecting for fungus resistance in vector populations to promote naturally diminished malaria vector competence.

Possible influence of Plasmodium/Trypanosoma co-infections on the vectorial capacity of Anopheles mosquitoes.

OBJECTIVE: In tropical Africa, trypanosomiasis is present in endemic areas with many other diseases including malaria. Because malaria vectors become more anthropo-zoophilic under the current insecticide pressure, they may be exposed to trypanosome parasites. By collecting mosquitoes in six study sites with distinct malaria infection prevalence and blood sample from cattle, we tried to assess the influence of malaria-trypanosomiasis co-endemicity on the vectorial capacity of Anopheles. RESULTS: Overall, all animal infections were due to Trypanosoma vivax (infection rates from 2.6 to 10.5%) in villages where the lowest Plasmodium prevalence were observed at the beginning of the study. An. gambiae s.l. displayed trophic preferences for human-animal hosts. Over 84 mosquitoes, only one was infected by Plasmodium falciparum (infection rate: 4.5%) in a site that displayed the highest prevalence at the beginning of the study. Thus, Anopheles could be exposed to Trypanosoma when they feed on infected animals. No Plasmodium infection was observed in the Trypanosoma-infected animals sites. This can be due to an interaction between both parasites as observed in mice and highlights the need of further studies considering Trypanosoma/Plasmodium mixed infections to better characterize the role of these infections in the dynamic of malaria transmission and the mechanisms involved.

Leucine-Rich Immune Factor APL1 Is Associated With Specific Modulation of Enteric Microbiome Taxa in the Asian Malaria Mosquito Anopheles stephensi.

The commensal gut microbiome is contained by the enteric epithelial barrier, but little is known about the degree of specificity of host immune barrier interactions for particular bacterial taxa. Here, we show that depletion of leucine-rich repeat immune factor APL1 in the Asian malaria mosquito Anopheles stephensi is associated with higher midgut abundance of just the family Enterobacteraceae, and not generalized dysbiosis of the microbiome. The effect is explained by the response of a narrow clade containing two main taxa related to Klebsiella and Cedecea. Analysis of field samples indicate that these two taxa are recurrent members of the wild Anopheles microbiome. Triangulation using sequence and functional data incriminated relatives of C. neteri and Cedecea NFIX57 as candidates for the Cedecea component, and K. michiganensis, K. oxytoca, and K.sp. LTGPAF-6F as candidates for the Klebsiella component. APL1 presence is associated with host ability to specifically constrain the abundance of a narrow microbiome clade of the Enterobacteraceae, and the immune factor may promote homeostasis of this clade in the enteric microbiome for host benefit.

Exposure of Anopheles mosquitoes to trypanosomes reduces reproductive fitness and enhances susceptibility to Plasmodium.

During a blood meal, female Anopheles mosquitoes are potentially exposed to diverse microbes in addition to the malaria parasite, Plasmodium. Human and animal African trypanosomiases are frequently co-endemic with malaria in Africa. It is not known whether exposure of Anopheles to trypanosomes influences their fitness or ability to transmit Plasmodium. Using cell and molecular biology approaches, we found that Trypanosoma brucei brucei parasites survive for at least 48h after infectious blood meal in the midgut of the major malaria vector, Anopheles coluzzii before being cleared. This transient survival of trypanosomes in the midgut is correlated with a dysbiosis, an alteration in the abundance of the enteric bacterial flora in Anopheles coluzzii. Using a developmental biology approach, we found that the presence of live trypanosomes in mosquito midguts also reduces their reproductive fitness, as it impairs the viability of laid eggs by affecting their hatching. Furthermore, we found that Anopheles exposure to trypanosomes enhances their vector competence for Plasmodium, as it increases their infection prevalence. A transcriptomic analysis revealed that expression of only two Anopheles immune genes are modulated during trypanosome exposure and that the increased susceptibility to Plasmodium was microbiome-dependent, while the reproductive fitness cost was dependent only on the presence of live trypanosomes but was microbiome independent. Taken together, these results demonstrate multiple effects upon Anopheles vector competence for Plasmodium caused by eukaryotic microbes interacting with the host and its microbiome, which may in turn have implications for malaria control strategies in co-endemic areas.

Gene copy number and function of the APL1 immune factor changed during Anopheles evolution.

BACKGROUND: The recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined. METHODS: The APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis. RESULTS: APL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus. CONCLUSIONS: APL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa.

Interaction of RNA viruses of the natural virome with the African malaria vector, Anopheles coluzzii.

Mosquitoes are colonized by a little-studied natural virome. Like the bacterial microbiome, the virome also probably influences the biology and immunity of mosquito vector populations, but tractable experimental models are lacking. We recently discovered two novel viruses in the virome of wild Anopheles and in colonies of the malaria vector Anopheles coluzzii: Anopheles C virus and Anopheles cypovirus. Here, we describe biological interactions between these two viruses and An. coluzzii mosquitoes. Viral abundance varies reproducibly during mosquito development. DNA forms of these viruses were not detected, and thus viral persistence is likely based on vertical transmission of RNA genomes. At least Anopheles C virus is vertically transmitted by an intraembryonic route. Relative abundance of the two viruses is inversely correlated in individual mosquitoes. One possible mechanism for this could be interactions with host immunity, and functional genomic analysis indicated differential influence of at least the Toll and JAK/STAT immune signaling pathways upon the viruses. The nonrandom distributions and interactions with host immunity suggest that these and other members of the natural virome may constitute a source of unrecognized heterogeneity in mosquito vector populations.

Dual role of the Anopheles coluzzii Venus Kinase Receptor in both larval growth and immunity.

Vector-borne diseases and especially malaria are responsible for more than half million deaths annually. The increase of insecticide resistance in wild populations of Anopheles malaria vectors emphasises the need for novel vector control strategies as well as for identifying novel vector targets. Venus kinase receptors (VKRs) constitute a Receptor Tyrosine Kinase (RTK) family only found in invertebrates. In this study we functionally characterized Anopheles VKR in the Gambiae complex member, Anopheles coluzzii. Results showed that Anopheles VKR can be activated by L-amino acids, with L-arginine as the most potent agonist. VKR was not required for the fecundity of A. coluzzii, in contrast to reports from other insects, but VKR function is required in both Anopheles males and females for development of larval progeny. Anopheles VKR function is also required for protection against infection by Plasmodium parasites, thus identifying a novel linkage between reproduction and immunity in Anopheles. The insect specificity of VKRs as well as the essential function for reproduction and immunity suggest that Anopheles VKR could be a potentially druggable target for novel vector control strategies.

Microbial Pre-exposure and Vectorial Competence of Anopheles Mosquitoes.

Anopheles female mosquitoes can transmit Plasmodium, the malaria parasite. During their aquatic life, wild Anopheles mosquito larvae are exposed to a huge diversity of microbes present in their breeding sites. Later, adult females often take successive blood meals that might also carry different micro-organisms, including parasites, bacteria, and viruses. Therefore, prior to Plasmodium ingestion, the mosquito biology could be modulated at different life stages by a suite of microbes present in larval breeding sites, as well as in the adult environment. In this article, we highlight several naturally relevant scenarios of Anopheles microbial pre-exposure that we assume might impact mosquito vectorial competence for the malaria parasite: (i) larval microbial exposures; (ii) protist co-infections; (iii) virus co-infections; and (iv) pathogenic bacteria co-infections. In addition, significant behavioral changes in African Anopheles vectors have been associated with increasing insecticide resistance. We discuss how these ethological modifications may also increase the repertoire of microbes to which mosquitoes could be exposed, and that might also influence their vectorial competence. Studying Plasmodium-Anopheles interactions in natural microbial environments would efficiently contribute to refining the transmission risks.

Correction: An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

[This corrects the article DOI: 10.1371/journal.ppat.1005306.].

Genetic Structure of a Local Population of the Anopheles gambiae Complex in Burkina Faso.

Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa. Population heterogeneities for ecological and behavioral attributes expand and stabilize malaria transmission over space and time, and populations may change in response to vector control, urbanization and other factors. There is a need for approaches to comprehensively describe the structure and characteristics of a sympatric local mosquito population, because incomplete knowledge of vector population composition may hinder control efforts. To this end, we used a genome-wide custom SNP typing array to analyze a population collection from a single geographic region in West Africa. The combination of sample depth (n = 456) and marker density (n = 1536) unambiguously resolved population subgroups, which were also compared for their relative susceptibility to natural genotypes of Plasmodium falciparum malaria. The population subgroups display fluctuating patterns of differentiation or sharing across the genome. Analysis of linkage disequilibrium identified 19 new candidate genes for association with underlying population divergence between sister taxa, A. coluzzii (M-form) and A. gambiae (S-form).

An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with broad specificity a rarity.

Association mapping by pooled sequencing identifies TOLL 11 as a protective factor against Plasmodium falciparum in Anopheles gambiae.

BACKGROUND: The genome-wide association study (GWAS) techniques that have been used for genetic mapping in other organisms have not been successfully applied to mosquitoes, which have genetic characteristics of high nucleotide diversity, low linkage disequilibrium, and complex population stratification that render population-based GWAS essentially unfeasible at realistic sample size and marker density. METHODS: We designed a novel mapping strategy for the mosquito system that combines the power of linkage mapping with the resolution afforded by genetic association. We established founder colonies from West Africa, controlled for diversity, linkage disequilibrium and population stratification. Colonies were challenged by feeding on the infectious stage of the human malaria parasite, Plasmodium falciparum, mosquitoes were phenotyped for parasite load, and DNA pools for phenotypically similar mosquitoes were Illumina sequenced. Phenotype-genotype mapping was carried out in two stages, coarse and fine. RESULTS: In the first mapping stage, pooled sequences were analysed genome-wide for intervals displaying relativereduction in diversity between phenotype pools, and candidate genomic loci were identified for influence upon parasite infection levels. In the second mapping stage, focused genotyping of SNPs from the first mapping stage was carried out in unpooled individual mosquitoes and replicates. The second stage confirmed significant SNPs in a locus encoding two Toll-family proteins. RNAi-mediated gene silencing and infection challenge revealed that TOLL 11 protects mosquitoes against P. falciparum infection. CONCLUSIONS: We present an efficient and cost-effective method for genetic mapping using natural variation segregating in defined recent Anopheles founder colonies, and demonstrate its applicability for mapping in a complex non-model genome. This approach is a practical and preferred alternative to population-based GWAS for first-pass mapping of phenotypes in Anopheles. This design should facilitate mapping of other traits involved in physiology, epidemiology, and behaviour.

The kdr-bearing haplotype and susceptibility to Plasmodium falciparum in Anopheles gambiae: genetic correlation and functional testing.

BACKGROUND: Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa. It is known that a large haplotype shared between An. gambiae and Anopheles coluzzii by introgression carries point mutations of the voltage-gated sodium channel gene para, including the L1014F kdr mutation associated with insensitivity to pyrethroid insecticides. Carriage of L1014F kdr is also correlated with higher susceptibility to infection with Plasmodium falciparum. However, the genetic mechanism and causative gene(s) underlying the parasite susceptibility phenotype are not known. METHODS: Mosquitoes from the wild Burkina Faso population were challenged by feeding on natural P. falciparum gametocytes. Oocyst infection phenotypes were determined and were tested for association with SNP genotypes. Candidate genes in the detected locus were prioritized and RNAi-mediated gene silencing was used to functionally test for gene effects on P. falciparum susceptibility. RESULTS: A genetic locus, Pfin6, was identified that influences infection levels of P. falciparum in mosquitoes. The locus segregates as a ~3 Mb haplotype carrying 65 predicted genes including the para gene. The haplotype carrying the kdr allele of para is linked to increased parasite infection prevalence, but many single nucleotide polymorphisms on the haplotype are also equally linked to the infection phenotype. Candidate genes in the haplotype were prioritized and functionally tested. Silencing of para did not influence P. falciparum infection, while silencing of a predicted immune gene, serine protease ClipC9, allowed development of significantly increased parasite numbers. CONCLUSIONS: Genetic variation influencing Plasmodium infection in wild Anopheles is linked to a natural ~3 megabase haplotype on chromosome 2L that carries the kdr allele of the para gene. Evidence suggests that para gene function does not directly influence parasite susceptibility, and the association of kdr with infection may be due to tight linkage of kdr with other gene(s) on the haplotype. Further work will be required to determine if ClipC9 influences the outcome of P. falciparum infection in nature, as well as to confirm the absence of a direct influence by para.

Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.

Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o'nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens.

Gαo is required for L-canavanine detection in Drosophila.

Taste is an essential sense for the survival of most organisms. In insects, taste is particularly important as it allows to detect and avoid ingesting many plant toxins, such as L-canavanine. We previously showed that L-canavanine is toxic for Drosophila melanogaster and that flies are able to detect this toxin in the food. L-canavanine is a ligand of DmXR, a variant G-protein coupled receptor (GPCR) belonging to the metabotropic glutamate receptor subfamily that is expressed in bitter-sensitive taste neurons of Drosophila. To transduce the signal intracellularly, GPCR activate heterotrimeric G proteins constituted of α, ß and γ subunits. The aim of this study was to identify which Gα protein was required for L-canavanine detection in Drosophila. By using a pharmacological approach, we first demonstrated that DmXR has the best coupling with Gαo protein subtype. Then, by using genetic, behavioral assays and electrophysiology, we found that Gαo47A is required in bitter-sensitive taste neurons for L-canavanine sensitivity. In conclusion, our study revealed that Gαo47A plays a crucial role in L-canavanine detection.

Anopheles gambiae pathogen susceptibility: the intersection of genetics, immunity and ecology.

Mosquitoes are the major arthropod vectors of human diseases such as malaria and viral encephalitis. However, each mosquito species does not transmit every pathogen, owing to reasons that include specific evolutionary histories, mosquito immune system structure, and ecology. Even a competent vector species for a pathogen displays a wide range of variation between individuals for pathogen susceptibility, and therefore efficiency of disease transmission. Understanding the molecular and genetic mechanisms that determine heterogeneities in transmission efficiency within a vector species could help elaborate new vector control strategies. This review discusses mechanisms of host-defense in Anopheles gambiae, and sources of genetic and ecological variation in the operation of these protective factors. Comparison is made between functional studies using Plasmodium or fungus, and we call attention to the limitations of generalizing gene phenotypes from experiments done in a single genetically simple colony.

Diverged alleles of the Anopheles gambiae leucine-rich repeat gene APL1A display distinct protective profiles against Plasmodium falciparum.

Functional studies have demonstrated a role for the Anopheles gambiae APL1A gene in resistance against the human malaria parasite, Plasmodium falciparum. Here, we exhaustively characterize the structure of the APL1 locus and show that three structurally different APL1A alleles segregate in the Ngousso colony. Genetic association combined with RNAi-mediated gene silencing revealed that APL1A alleles display distinct protective profiles against P. falciparum. One APL1A allele is sufficient to explain the protective phenotype of APL1A observed in silencing experiments. Epitope-tagged APL1A isoforms expressed in an in vitro hemocyte-like cell system showed that under assay conditions, the most protective APL1A isoform (APL1A(2)) localizes within large cytoplasmic vesicles, is not constitutively secreted, and forms only one protein complex, while a less protective isoform (APL1A(1)) is constitutively secreted in at least two protein complexes. The tested alleles are identical to natural variants in the wild A. gambiae population, suggesting that APL1A genetic variation could be a factor underlying natural heterogeneity of vector susceptibility to P. falciparum.

Anopheles gambiae PRS1 modulates Plasmodium development at both midgut and salivary gland steps.

BACKGROUND: Invasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches. METHODOLOGY/PRINCIPAL FINDINGS: PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues. CONCLUSIONS/SIGNIFICANCE: While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.