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Melanins are structurally complex pigments produced by organisms in all domains of life. In insects, melanins are essential for survival and have key roles in cuticle sclerotization, wound healing and innate immunity. In this study, we used a diverse set of molecular, biochemical, and imaging approaches to characterize mosquito melanin involved in innate immune defense (melanotic capsules). We observed that melanotic capsules enclosing Plasmodium berghei ookinetes were composed of an acid-resistant and highly hydrophobic material with granular appearance, which are characteristic properties of melanins. Spectroscopical analyses reveal chemical signatures of eumelanins and pheomelanin. Furthermore, we identified a set of 14 acid-resistant mosquito proteins embedded within the melanin matrix possibly related to an anti-Plasmodium response. Among these, AgMESH, a mucin-related protein highly conserved among insects that is associated with the midgut brush border microvilli proteome of Anopheles gambiae and A. albimanus. AgMESH gene silencing in mosquitos was associated with reduced Plasmodium parasite infection, compromised integrity of the peritrophic matrix, and inability to synthesize a dityrosine network. Our results provide a new approach to study aspects of insect melanogenesis that revealed proteins associated with melanotic capsule, one of which was strongly implicated in the stabilization of the peritrophic matrix and pathogenesis of Plasmodium spp. mosquito infection. Given the conservation of AgMESH among disease-transmitting insect vector species, future analysis of this protein could provide fertile ground for the identification of strategies that block transmission of vector borne diseases to humans.Significance StatementMalaria is a parasitic disease transmitted by mosquito bites. Here, we adapt methodologies to study fungal melanogenesis to explore the melanin-based immune response of Anopheles gambiae against malaria parasites. We reveal that melanotic capsules against Plasmodium are composed of pheomelanin and eumelanin. We demonstrate that melanin-encapsulated Plasmodium is associated to acid-resistant mosquito gut proteins and identify several putative factors of the melanin-mediated immunity. Disruption of AgMESH, a surface-associated protein conserved among other mosquito vectors, demonstrates its ability to impaired formation of the dityrosine network and peritrophic matrix compromising parasite development within the mosquito gut. Our study provides a new approach to investigate the melanin-based defense mechanism in insects and identified a potential host molecule for developing novel universal vector-control schemes.

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin’s broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate—the most widely used herbicide globally—inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate’s mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation–reduction balance. Overall, these findings suggest that glyphosate’s environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations., Glyphosate, the most commonly used herbicide in the world, inhibits the production of melanin. Melanin is an important pigment and a key component of the insect immune system; this study shows that glyphosate weakens insects’ melanin-based immune system and makes them more vulnerable to infections, including with the human malaria parasite Plasmodium falciparum.

Background Vector control is critical in reducing the disease burden caused by mosquitoes, and insecticides are an effective tool to control vector populations. Resistance to common insecticides is now widespread, and novel classes of insecticides are needed. In previous work, we described the mosquitocidal activity of Chromobacterium sp. Panama (C.sp_P), a bacterium found in association with mosquitoes in natural populations. In the current work, we further explored the effects of exposure to the bacterium on mosquito fitness and mosquito physiology. Results We found that C.sp_P has mosquitocidal activity against a broad range of mosquito taxa. When exposed to C.sp_P as adults, female An. gambiae suffered reduced longevity, but experienced no change in fecundity. The offspring of these females, however, had higher mortality as larvae and were slower to develop compared to offspring of control females. We also found that the mosquitocidal activity of C.sp_P was retained after removal of live cells from biofilm culture media, suggesting the bacteria secrete mosquitocidal compound(s) into the media during growth. Exposure to this cell-free C.sp_P-conditioned media caused female midgut transcriptional changes comprising detoxification, xenobiotic response, and stress response genes, suggesting the physiological response to C.sp_P is similar to that of insecticide exposure. Finally, we found that multiple members of the Chromobacterium genus had mosquitocidal activity, but this activity was highest in mosquitoes treated with C.sp_P. Conclusions Our findings suggest that C.sp_P produces factor(s) with strong effects on mosquito longevity and fitness, which may be of interest for mosquitocide development. More generally, they indicate that further exploration of mosquito-associated and environmental microbes for novel insecticidal compounds or biocontrol agents is warranted. Electronic supplementary material The online version of this article (10.1186/s13071-018-2822-8) contains supplementary material, which is available to authorized users.

CRISPR-mediated disruption of a mosquito gene, mosGILT, impairs ovarian development and vitellogenesis, resulting in enhanced anti-Plasmodium immunity in the malaria vector Anopheles gambiae., Plasmodium infection in Anopheles is influenced by mosquito-derived factors. We previously showed that a protein in saliva from infected Anopheles, mosquito gamma-interferon–inducible lysosomal thiol reductase (mosGILT), inhibits the ability of sporozoites to traverse cells and readily establish infection of the vertebrate host. To determine whether mosGILT influences Plasmodium within the mosquito, we generated Anopheles gambiae mosquitoes carrying mosaic mutations in the mosGILT gene using CRISPR/CRISPR associated protein 9 (Cas9). Here, we show that female mosaic mosGILT mutant mosquitoes display defects in ovarian development and refractoriness to Plasmodium. Following infection by either Plasmodium berghei or Plasmodium falciparum, mutant mosquitoes have significantly reduced oocyst numbers as a result of increased thioester-containing protein 1 (TEP1)–dependent parasite killing. Expression of vitellogenin (Vg), the major yolk protein that can reduce the parasite-killing efficiency of TEP1, is severely impaired in mutant mosquitoes. MosGILT is a mosquito factor that is essential for ovarian development and indirectly protects both human and rodent Plasmodium species from mosquito immunity., Graphical Abstract

Antibodies to AgTRIO, a mosquito salivary protein, partially reduce the initial Plasmodium burden in mice. We therefore silenced AgTRIO in mosquitoes and determined the relative contribution of AgTRIO to the ability of Anopheles gambiae to transmit Plasmodium berghei to mice. RNA interference-mediated silencing of AgTRIO inA. gambiae resulted in a 60% reduction in AgTRIO expression. The decrease in AgTRIO expression did not alter the burden of Plasmodium sporozoites in mosquito salivary glands. When experimentally injected into mice, sporozoites from AgTRIO-silenced mosquitoes colonized the liver less effectively than sporozoites from control mosquitoes. Silencing of AgTRIO did not decrease the infectivity of sporozoites in vitro or influence the expression of genes associated with Plasmodium cell adhesion or traversal activity. AgTRIO decreased the expression of proinflammation cytokines by splenocytes in vitro. Moreover, in vivo, AgTRIO decreased the expression of TNF-α when coinjected with sporozoites into the skin and there was more TNF-α expression at the bite site of AgTRIO knockdown mosquitoes than at the bite site of control mosquitoes. AgTRIO therefore influences the local environment in the vertebrate host, which facilitates Plasmodium sporozoite infection in mice.

Malaria, caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes, represents a major threat to human health. Plasmodium’s infection cycle in the Anopheles vector is critical for transmission of the parasite between humans. The midgut-stage bottleneck of infection is largely imposed by the mosquito’s innate immune system. microRNAs (miRNAs, small noncoding RNAs that bind to target RNAs to regulate gene expression) are also involved in regulating immunity and the anti-Plasmodium defense in mosquitoes. Here, we characterized the mosquito’s miRNA responses to Plasmodium infection using an improved crosslinking and immunoprecipitation (CLIP) method, termed covalent ligation of endogenous Argonaute-bound RNAs (CLEAR)-CLIP. Three candidate miRNAs’ influence on P. falciparum infection and midgut microbiota was studied through transgenically expressed miRNA sponges (miR-SPs) in midgut and fat body tissues. MiR-SPs mediated conditional depletion of aga-miR-14 or aga-miR-305, but not aga-miR-8, increased mosquito resistance to both P. falciparum and P. berghei infection, and enhanced the mosquitoes’ antibacterial defenses. Transcriptome analysis revealed that depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes in mosquito midguts. The overall fitness cost of conditionally expressed miR-SPs was low, with only one of eight fitness parameters being adversely affected. Taken together, our results demonstrate that targeting mosquito miRNA by conditional expression of miR-SPs may have potential for the development of malaria control through genetically engineered mosquitoes., Author summary Malaria is caused by the Plasmodium parasite that is transmitted by Anopheles mosquitoes. The mosquito’s innate immune system plays an important role in controlling parasite infection. We have identified mosquito microRNAs (miRNAs) that are involved in regulating mosquito immunity to parasite infection. Transgenic mosquitoes that deplete the immunity-related miRNAs aga-miR-14 or aga-miR-305 through miRNA sponges, show increased resistance to both human and rodent parasite infection, and enhanced antibacterial defenses. Depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes, and the overall fitness cost of transgenic mosquitoes upon depletion of aga-miR-14 or aga-miR-305 was negligible. We show that targeting mosquito miRNA by transgenic expression of miRNA sponges may have potential for the development of malaria control through genetically engineered mosquitoes.

Plasmodium relies on numerous agonists during its journey through the mosquito vector, and these agonists represent potent targets for transmission-blocking by either inhibiting or interfering with them pre- or post-transcriptionally. The recently developed CRISPR/Cas9-based genome editing tools for Anopheles mosquitoes provide new and promising opportunities for the study of agonist function and for developing malaria control strategies through gene deletion to achieve complete agonist inactivation. Here we have established a modified CRISPR/Cas9 gene editing procedure for the malaria vector Anopheles gambiae, and studied the effect of inactivating the fibrinogen-related protein 1 (FREP1) gene on the mosquito’s susceptibility to Plasmodium and on mosquito fitness. FREP1 knockout mutants developed into adult mosquitoes that showed profound suppression of infection with both human and rodent malaria parasites at the oocyst and sporozoite stages. FREP1 inactivation, however, resulted in fitness costs including a significantly lower blood-feeding propensity, fecundity and egg hatching rate, a retarded pupation time, and reduced longevity after a blood meal., Author summary The causative agent of malaria, Plasmodium, has to complete a complex infection cycle in the Anopheles gambiae mosquito vector in order to reach the salivary gland from where it can be transmitted to a human host. The parasite’s development in the mosquito relies on numerous host factors (agonists), and their inhibition or inactivation can thereby result in suppression of infection and consequently malaria transmission. The recently developed CRISPR/Cas9-based genome editing tools for Anopheles mosquitoes provide new and promising opportunities to delete (inactivate) Plasmodium agonists to better understand their function and for blocking malaria transmission. Here we have established a modified CRISPR/Cas9 genome editing technique for malaria vector A. gambiae mosquitoes. Through this approach we have inactivated the fibrinogen-related protein 1 (FREP1) gene, via CRISPR/Cas9 genome editing, and the impact of this manipulation on the mosquito’s susceptibility to Plasmodium and on mosquito fitness. FREP1 knockout mutants showed a profound suppression of infection with both human and rodent malaria parasites, while it also resulted in fitness costs: a significantly lower blood-feeding propensity, fecundity and egg hatching rate, and a retarded larval development and pupation time, and reduced longevity after a blood meal.

Malaria parasite ookinetes must traverse the vector mosquito midgut epithelium to transform into sporozoite-producing oocysts. The Anopheles innate immune system is a key regulator of this process, thereby determining vector competence and disease transmission. The role of Anopheles innate immunity factors as agonists or antagonists of malaria parasite infection has been previously determined using specific single Anopheles - Plasmodium species combinations. Here we show that the two C-type lectins CTL4 and CTLMA2 exert differential agonistic and antagonistic regulation of parasite killing in African and South American Anopheles species. The C-type lectins regulate both parasite melanization and lysis through independent mechanisms, and their implication in parasite melanization is dependent on infection intensity rather than mosquito-parasite species combination. We show that the leucine-rich repeat protein LRIM1 acts as an antagonist on the development of Plasmodium ookinetes and as a regulator of oocyst size and sporozoite production in the South American mosquito Anopheles albimanus . Our findings explain the rare observation of human Plasmodium falciparum melanization and define a key factor mediating the poor vector competence of Anopheles albimanus for Plasmodium berghei and Plasmodium falciparum . IMPORTANCE Malaria, one of the world’s deadliest diseases, is caused by Plasmodium parasites that are vectored to humans by the bite of Anopheles mosquitoes. The mosquito’s innate immune system is actively engaged in suppressing Plasmodium infection. Studies on mosquito immunity revealed multiple factors that act as either facilitators or inhibitors of Plasmodium infection, but these findings were mostly based on single Anopheles - Plasmodium species combinations, not taking into account the diversity of mosquito and parasite species. We show that the functions of CTL4 and CTLMA2 have diverged in different vector species and can be both agonistic and antagonistic for Plasmodium infection. Their protection against parasite melanization in Anopheles gambiae is dependent on infection intensity, rather than the mosquito-parasite combination. Importantly, we describe for the first time how LRIM1 plays an essential role in Plasmodium infection of Anopheles albimanus , suggesting it is a key regulator of the poor vector competence of this species.

Mosquitoes have a multifaceted innate immune system that is actively engaged in warding off various pathogens, including the protozoan malaria parasite Plasmodium . Various immune signaling pathways and effectors have been shown to mediate a certain degree of defense specificity against different Plasmodium species. A key pattern recognition receptor of the Anopheles gambiae immune system is the fibrinogen domain-containing immunolectin FBN9, which has been shown to be transcriptonally induced by Plasmodium infection, and to mediate defense against both rodent and human malaria parasites and bacteria. Here we have further studied the defense specificity of FBN9 using a transgenic approach, in which FBN9 is overexpressed in the fat body tissue after a blood meal through a vitellogenin promoter. Interestingly, the Vg-FBN9 transgenic mosquitoes showed increased resistance only to the rodent parasite P. berghei, and not to the human parasite P. falciparum , pointing to differences in the mosquito's defense mechanisms against the two parasite species. The Vg-FBN9 transgenic mosquitoes were also more resistant to infection with both Gram-positive and Gram-negative bacteria and showed increased longevity when infected with P. berghei . Our study points to the importance of both experimentally depleting and enriching candidate anti- Plasmodium effectors in functional studies in order to ascertain their suitability for the development of transgenic mosquito–based malaria control strategies.

Malaria parasite transmission depends upon the successful development of Plasmodium in its Anopheles mosquito vector. The mosquito's innate immune system constitutes a major bottleneck for parasite population growth. We show here that in Anopheles gambiae, the midgut-specific transcription factor Caudal acts as a negative regulator in the Imd pathway-mediated immune response against the human malaria parasite Plasmodium falciparum. Caudal also modulates the mosquito midgut bacterial flora. RNAi-mediated silencing of Caudal enhanced the mosquito's resistance to bacterial infections and increased the transcriptional abundance of key immune effector genes. Interestingly, Caudal's silencing resulted in an increased lifespan of the mosquito, while it impaired reproductive fitness with respect to egg laying and hatching.

Whereas studies have extensively examined the ability of bacteria to influence Plasmodium infection in the mosquito, the tripartite interactions between non-entomopathogenic fungi, mosquitoes and Plasmodium parasites remain largely uncharacterized. Here we report the isolation of a common mosquito-associated ascomycete fungus, Penicillium chrysogenum, from the midgut of field-caught Anopheles mosquitoes. Although the presence of Pe. chrysogenum in the Anopheles gambiae midgut does not affect mosquito survival, it renders the mosquito significantly more susceptible to Plasmodium infection through a secreted heat-stable factor. We further provide evidence that the mechanism of the fungus-mediated modulation of mosquito susceptibility to Plasmodium involves an upregulation of the insect’s ornithine decarboxylase gene, which sequesters arginine for polyamine biosynthesis. Arginine plays an important role in the mosquito’s anti-Plasmodium defense as a substrate of nitric oxide production and its availability therefore has a direct impact on the mosquito’s susceptibility to the parasite. While this type of immunomodulatory mechanism has already been demonstrated in other host-pathogen interaction systems, this is the first report of a mosquito-associated fungus that can suppress the mosquito’s innate immune system in a way that would favor Plasmodium infection and possibly malaria transmission.

Gram-negative bacteria-binding proteins (GNBPs) are pattern recognition receptors which contribute to the defensive response against Plasmodium infection in Anopheles. We have characterized the GNBP gene family in Anopheles gambiae at the molecular level, and show that they are functionally diverse components of the A. gambiae innate immune system. GNBPB4 is a major factor in the defence against a broad range of pathogens, while the other GNBPs have narrower defence specificities. GNBPB4 is associated with the regulation of immune signalling pathways and was found to interact with the Gram-negative Escherichia coli and weakly co-localized with Plasmodium berghei ookinetes in the mosquito midgut epithelium.

The molecular biology of disease vectors, particularly mosquitoes, has experienced a remarkable progress in the past two decades. This is mainly attributed to methodological advances and the emerging genome sequences of vector species, which have brought experimental biology to an unprecedented level. It is now possible to determine the entire transcriptome of Anopheles gambiae at a variety of conditions, with a low per-gene effort and cost. Proteomic profiles can be generated for as small samples as the hemolymph, and transient reverse genetic and stable germ line based transgenic analyses can be performed to analyze gene function. High throughput screening for receptors and ligands can be used to characterize interactions between vectors and pathogens. At the current breathtaking rates of data production it is essential to question and evaluate the relevance of laboratory infection models to the real disease transmission systems. The majority of scientific discoveries in mosquito molecular biology have been based on highly inbred laboratory strains and rodent malaria parasite infection models, which may differ substantially to their counterparts that transmit human malaria in the field. This review addresses the recent advances in high throughput transcription analyses of Anopheles responses to infection, and discusses considerations for the use of laboratory malaria infection models.

Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae, and FREP1 is important for Plasmodium berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a blood meal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito-expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission.

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito's vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies., Author Summary The infectious agents that cause malaria and dengue are transmitted by Anopheles and Aedes mosquitoes, respectively. Bacteria found in the mosquito midgut have the potential to dramatically affect the susceptibility of the mosquito vector to the malaria parasite and dengue virus. In this work, we investigate one such microbe, Chromobacterium sp. (Csp_P), a bacterium isolated from a field-caught Aedes aegypti mosquito. We show that Csp_P can effectively colonize the midguts of Anopheles gambiae and Aedes aegypti mosquitoes and can, when ingested by the mosquito, significantly reduce the mosquito's susceptibility to infection with the malaria parasite and dengue virus. We also show that exposure to, and ingestion of, Csp_P can reduce the lifespan of larval and adult mosquitoes, respectively. We show that Csp_P has anti-Plasmodium and anti-dengue activity independent of the mosquito, suggesting that the bacterium secretes metabolites that could potentially be exploited to prevent disease transmission or to treat infection.

The malarial parasite Plasmodium must complete a complex lifecycle in its Anopheles mosquito host, the main vector for Plasmodium. The mosquito resists infection with the human malarial parasite P. falciparum by engaging the NF-κB immune signaling pathway, IMD. Here we show that the conserved transcriptional mediators Kto and Skd are involved in the regulation of the mosquito IMD pathway. RNAi-mediated depletion of Kto and Skd in the Anopheles gambiae cell line L5-3 resulted in a decrease in the transcript abundance of Cec1, which is controlled by the IMD pathway. Silencing the two genes also resulted in an increased susceptibility of the mosquito to bacterial and Plasmodium falciparum infection, but not to infection with the rodent malaria parasite P. berghei. We also showed that Kto and Skd are not transcriptional co-activators of Rel2 or other key factors of the IMD pathway; however, they participate in the regulation of the IMD pathway, which is crucial for the mosquito's defense against P. falciparum.

Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito 99% resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development prior to invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species.

Malaria-transmitting mosquitoes are continuously exposed to microbes, including their midgut microbiota. This naturally acquired microbial flora can modulate the mosquito's vectorial capacity by inhibiting the development of Plasmodium and other human pathogens through an unknown mechanism. We have undertaken a comprehensive functional genomic approach to elucidate the molecular interplay between the bacterial co-infection and the development of the human malaria parasite Plasmodium falciparum in its natural vector Anopheles gambiae. Global transcription profiling of septic and aseptic mosquitoes identified a significant subset of immune genes that were mostly up-regulated by the mosquito's microbial flora, including several anti-Plasmodium factors. Microbe-free aseptic mosquitoes displayed an increased susceptibility to Plasmodium infection while co-feeding mosquitoes with bacteria and P. falciparum gametocytes resulted in lower than normal infection levels. Infection analyses suggest the bacteria-mediated anti-Plasmodium effect is mediated by the mosquitoes' antimicrobial immune responses, plausibly through activation of basal immunity. We show that the microbiota can modulate the anti-Plasmodium effects of some immune genes. In sum, the microbiota plays an essential role in modulating the mosquito's capacity to sustain Plasmodium infection., Author Summary The Anopheles gambiae mosquito that transmits the malaria-causing parasite Plasmodium has an intestinal bacterial flora, or microbiota, which comprises a variety of species. Elimination of this microbiota with antibiotic treatment will render the Anopheles mosquito more susceptible to Plasmodium infection. In this study we show that these bacteria can inhibit the infection of the mosquito with the human malaria parasite Plasmodium falciparum through a mechanism that involves the mosquito's immune system. Our study suggests that the microbial flora of mosquitoes is stimulating a basal immune activity, which comprises several factors with known anti-Plasmodium activity. The same immune factors that are needed to control the mosquito's microbiota are also defending against the malaria parasite Plasmodium. This complex interplay among the mosquito's microbiota, the innate immune system, and the Plasmodium parasite may have significant implications for the transmission of malaria in the field where the bacterial exposure of mosquitoes may differ greatly between ecological niches.

The fibrinogen-related protein family (FREP, also known as FBN) is an evolutionarily conserved immune gene family found in mammals and invertebrates. It is the largest pattern recognition receptor gene family in Anopheles gambiae, with as many as 59 putative members, while the Drosophila melanogaster genome has only 14 known FREP members. Our sequence and phylogenetic analysis suggest that this remarkable gene expansion in the mosquito is the result of tandem duplication of the fibrinogen domain. We found that the majority of the FREP genes displayed immune-responsive transcription after challenge with bacteria, fungi, or Plasmodium, and these expression patterns correlated strongly with gene phylogeny and chromosomal location. Using RNAi-mediated gene-silencing assays, we further demonstrated that some FREP members are essential factors of the mosquito innate immune system that are required for maintaining immune homeostasis, and members of this family have complementary and synergistic functions. One of the most potent anti-Plasmodium FREP proteins, FBN9, was found to interact with both Gram-negative and Gram-positive bacteria and strongly co-localized with both rodent and human malaria parasites in the mosquito midgut epithelium, suggesting that its defensive activity involves direct interaction with the pathogen. Interestingly, FBN9 formed dimers that bound to the bacterial surfaces with different affinities. Our findings indicate that the A. gambiae FREP gene family plays a central role in the mosquito innate immune system and provides an expanded pattern recognition and anti-microbial defense repertoire.

Background The midgut of hematophagous insects, such as disease transmitting mosquitoes, carries out a variety of essential functions that mostly relate to blood feeding. The midgut of the female malaria vector mosquito Anopheles gambiae is a major site of interactions between the parasite and the vector. Distinct compartments and cell types of the midgut tissue carry out specific functions and vector borne pathogens interact and infect different parts of the midgut. Results A microarray based global gene expression approach was used to compare transcript abundance in the four major female midgut compartments (cardia, anterior, anterior part of posterior and posterior part of posterior midgut) and between the male and female Anopheles gambiae midgut. Major differences between the female and male midgut gene expression relate to digestive processes and immunity. Each compartment has a distinct gene function profile with the posterior midgut expressing digestive enzyme genes and the cardia and anterior midgut expressing high levels of antimicrobial peptide and other immune gene transcripts. Interestingly, the cardia expressed several known anti-Plasmodium factors. A parallel peptidomic analysis of the cardia identified known mosquito antimicrobial peptides as well as several putative short secreted peptides that are likely to represent novel antimicrobial factors. Conclusion The A. gambiae sex specific midgut and female midgut compartment specific transcriptomes correlates with their known functions. The significantly greater functional diversity of the female midgut relate to hematophagy that is associated with digestion and nutrition uptake as well as exposes it to a variety of pathogens, and promotes growth of its endogenous microbial flora. The strikingly high proportion of immunity related factors in the cardia tissue most likely serves the function to increase sterility of ingested sugar and blood. A detailed characterization of the functional specificities of the female mosquito midgut and its various compartments can greatly contribute to our understanding of its role in disease transmission and generate the necessary tools for the development of malaria control strategies.

Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti ( Aa ), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa , the malaria vector Anopheles gambiae ( Ag ), and the fruit fly Drosophila melanogaster ( Dm ). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.

Activation of the insect innate immune system is dependent on a limited number of pattern recognition receptors (PRRs) capable of interacting with pathogen-associated molecular pattern. Here we report a novel role of an alternatively spliced hypervariable immunoglobulin domain-encoding gene, Dscam, in generating a broad range of PRRs implicated in immune defense in the malaria vector Anopheles gambiae. The mosquito Down syndrome cell adhesion molecule gene, AgDscam, has a complex genome organization with 101 exons that can produce over 31,000 potential alternative splice forms with different combinations of adhesive domains and interaction specificities. AgDscam responds to infection by producing pathogen challenge-specific splice form repertoires. Transient silencing of AgDscam compromises the mosquito's resistance to infections with bacteria and the malaria parasite Plasmodium. AgDscam is mediating phagocytosis of bacteria with which it can associate and defend against in a splice form–specific manner. AgDscam is a hypervariable PRR of the A. gambiae innate immune system., AgDscam has 101 exons that can produce more than 31,000 potential alternative splice forms. Specific splice forms produced in response to infection by a given pathogen contribute to the mosquito's resistance against pathogens.

The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasite's ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum., Author Summary The immune response of the mosquito vector of Plasmodium has proven to possess powerful anti-Plasmodium defense capabilities. As the major regulators of these immune responses, signaling pathways, particularly the Imd pathway which seems especially capable of eliminating malaria parasites, have become attractive candidates targets for malaria-control interventions. Although the general anti-parasitic activity of the Imd pathway has been established, the particular components of the pathway involved and the physiological conditions under which the pathway is capable of limiting infection are mostly unknown. Awareness of these major players and conditions is crucial for adapting the Imd pathway into an intervention strategy. We report that while several members of the Imd pathway are critical for such a response, others are dispensable. We also show that timing of the response with regard to infection and intensity of infection exposure both influence the effectiveness of an Imd-derived anti-Plasmodium response while the status of the gut flora does not. Taken together, this data lays the essential groundwork for effective intervention based on manipulation of this pathway that can severely limit mosquito infection with human malaria parasites.

Immune responses mounted by the malaria vector Anopheles gambiae are largely regulated by the Toll and Imd (immune deficiency) pathways via the NF-kappaB transcription factors Rel1 and Rel2, which are controlled by the negative regulators Cactus and Caspar, respectively. Rel1- and Rel2-dependent transcription in A. gambiae has been shown to be particularly critical to the mosquito's ability to manage infection with the rodent malaria parasite Plasmodium berghei. Using RNA interference to deplete the negative regulators of these pathways, we found that Rel2 controls resistance of A. gambiae to the human malaria parasite Plasmodium falciparum, whereas Rel 1 activation reduced infection levels. The universal relevance of this defense system across Anopheles species was established by showing that caspar silencing also prevents the development of P. falciparum in the major malaria vectors of Asia and South America, A. stephensi and A. albimanus, respectively. Parallel studies suggest that while Imd pathway activation is most effective against P. falciparum, the Toll pathway is most efficient against P. berghei, highlighting a significant discrepancy between the human pathogen and its rodent model. High throughput gene expression analyses identified a plethora of genes regulated by the activation of the two Rel factors and revealed that the Toll pathway played a more diverse role in mosquito biology than the Imd pathway, which was more immunity-specific. Further analyses of key anti-Plasmodium factors suggest they may be responsible for the Imd pathway–mediated resistance phenotype. Additionally, we found that the fitness cost caused by Rel2 activation through caspar gene silencing was undetectable in sugar-fed, blood-fed, and P. falciparum-infected female A. gambiae, while activation of the Toll pathway's Rel1 had a major impact. This study describes for the first time a single gene that influences an immune mechanism that is able to abort development of P. falciparum in Anopheline species. Further, this study addresses aspects of the molecular, evolutionary, and physiological consequences of the observed phenotype. These findings have implications for malaria control since broad-spectrum immune activation in diverse anopheline species offers a viable and strategic approach to develop novel malaria control methods worldwide., Author Summary The relationship between malaria parasites and the mosquitoes that transmit them to humans comprises complex molecular interactions including mosquito immune responses. Anopheles can mount potent anti-Plasmodium immune responses; we show here that the gene caspar, which encodes a negative regulator of the immune signaling pathway Imd, controls mosquito resistance to the human malaria parasite. Silencing of this Imd pathway regulator results in complete resistance to human Plasmodium in three divergent Anopheline malaria vector species, yet does not cause complete resistance to a rodent Plasmodium species, indicating conservation of defense function among mosquito but not diverse parasite species. We also identify a panel of genes that are transcriptionally regulated by silencing of the caspar gene. Some of these genes contribute directly to parasite resistance. Finally, we show that the transient immune activation that renders mosquitoes resistant to the human malaria parasite has little to no effect on mosquito fitness as a measure of survival or fecundity under laboratory conditions. In sum, this study shows that the mosquito's immune pathway, Imd, can regulate resistance to Plasmodium through immune responses that entail several known anti-Plasmodium genes.

Transmission of malaria is dependent on the successful completion of the Plasmodium lifecycle in the Anopheles vector. Major obstacles are encountered in the midgut tissue, where most parasites are killed by the mosquito's immune system. In the present study, DNA microarray analyses have been used to compare Anopheles gambiae responses to invasion of the midgut epithelium by the ookinete stage of the human pathogen Plasmodium falciparum and the rodent experimental model pathogen P. berghei. Invasion by P. berghei had a more profound impact on the mosquito transcriptome, including a variety of functional gene classes, while P. falciparum elicited a broader immune response at the gene transcript level. Ingestion of human malaria-infected blood lacking invasive ookinetes also induced a variety of immune genes, including several anti-Plasmodium factors. Twelve selected genes were assessed for effect on infection with both parasite species and bacteria using RNAi gene silencing assays, and seven of these genes were found to influence mosquito resistance to both parasite species. An MD2-like receptor, AgMDL1, and an immunolectin, FBN39, showed specificity in regulating only resistance to P. falciparum, while the antimicrobial peptide gambicin and a novel putative short secreted peptide, IRSP5, were more specific for defense against the rodent parasite P. berghei. While all the genes that affected Plasmodium development also influenced mosquito resistance to bacterial infection, four of the antimicrobial genes had no effect on Plasmodium development. Our study shows that the impact of P. falciparum and P. berghei infection on A. gambiae biology at the gene transcript level is quite diverse, and the defense against the two Plasmodium species is mediated by antimicrobial factors with both universal and Plasmodium-species specific activities. Furthermore, our data indicate that the mosquito is capable of sensing infected blood constituents in the absence of invading ookinetes, thereby inducing anti-Plasmodium immune responses., Synopsis The malarial parasite Plasmodium has to traverse the gut wall of the Anopheles mosquito in order to complete its lifecycle and to be transmitted between hosts. At the midgut stage of infection, the mosquito activates immune responses to eliminate most invading parasites. The features of these immune responses are not very well understood and have mainly been examined using the rodent parasite model P. berghei. Here the authors investigated the relationship between the Anopheles gambiae responses against the human pathogen P. falciparum, the rodent parasite P. berghei, and bacterial infections, at both the gene expression and functional levels. The mosquito responses against these pathogens were quite diverse, and the defense against the two malaria parasite species involved both common and species-specific components. Malaria-infected blood was sufficient to activate anti-Plasmodium immune responses, even in the absence of midgut invasion. Through this mechanism, the mosquito can initiate its defense against Plasmodium prior to invasion of the gut. Mosquito genes that could negatively influence Plasmodium development were also capable of regulating the resistance to bacterial infection, but several of the antibacterial genes had no effect on Plasmodium; thus, the mosquito apparently utilizes its antibacterial defense systems against the malaria parasite.

Background Plasmodium infection has been shown to compromise the fitness of the mosquito vector, reducing its fecundity and longevity. However, from an evolutionary perspective, the impact of Plasmodium infection as a selective pressure on the mosquito is largely unknown. Results In the present study we have addressed the effect of a continuous Plasmodium berghei infection on the resistance to infection and global gene expression in Anopheles gambiae. Exposure of A. gambiae to P. berghei-infected blood and infection for 16 generations resulted in a decreased susceptibility to infection, altered constitutive expression levels for approximately 2.4% of the mosquito's total transcriptome and a lower basal level of immune genes expression, including several anti-Plasmodium factors. The infection-responsiveness for several defense genes was elevated in the P. berghei exposed mosquito colonies. Conclusion Our study establishes the existence of a selective pressure exerted by the parasite P. berghei on the malaria vector A. gambiae that results in a decreased permissiveness to infection and changes in the mosquito transcriptome regulation that suggest a decreased constitutive immune gene activity but a more potent immune response upon Plasmodium challenge.