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Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito

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

The malaria parasite replicates asexually in the red blood cells of its vertebrate host employing epigenetic mechanisms to regulate gene expression in response to changes in its environment. We used chromatin immunoprecipitation followed by sequencing in conjunction with RNA sequencing to create an epigenomic and transcriptomic map of the developmental transition from asexual blood stages to male and female gametocytes and to ookinetes in the rodent malaria parasite Plasmodium berghei. Across the developmental stages examined, heterochromatin protein 1 associates with variantly expressed gene families localised at subtelomeric regions and variant gene expression based on heterochromatic silencing is observed only in some genes. Conversely, the euchromatin mark histone 3 lysine 9 acetylation (H3K9ac) is abundant in non-heterochromatic regions across all developmental stages. H3K9ac presents a distinct pattern of enrichment around the start codon of ribosomal protein genes in all stages but male gametocytes. Additionally, H3K9ac occupancy positively correlates with transcript abundance in all stages but female gametocytes suggesting that transcription in this stage is independent of H3K9ac levels. This finding together with known mRNA repression in female gametocytes suggests a multilayered mechanism operating in female gametocytes in preparation for fertilization and zygote development, coinciding with parasite transition from host to vector.

Significance Malaria is transmitted among humans through mosquito bites. Here, we characterize a protein found on the surface of mosquito stages of malaria parasites and reveal that it serves to evade the mosquito immune system and ensure disease transmission. Neutralization of PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43), either by eliminating it from the parasite genome or by preincubating parasites with antibodies that bind to the PIMMS43 protein, inhibits mosquito infection with malaria parasites. Differences in PIMMS43 detected between African malaria parasite populations suggest that these populations have adapted for transmission by different mosquito vectors that are also differentially distributed across the continent. We conclude that targeting PIMMS43 can block malaria parasites inside mosquitoes before they can infect humans., After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito–parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.

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

Inhibiting transmission of Plasmodium is an essential strategy in malaria eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. Lack of knowledge of the mechanisms underlying fertilization have been a hindrance in the development of transmission-blocking interventions. Here we describe a protein disulphide isomerase essential for malarial transmission (PDI-Trans/PBANKA_0820300) to the mosquito. We show that PDI-Trans activity is male-specific, surface-expressed, essential for fertilization/transmission, and exhibits disulphide isomerase activity which is up-regulated post-gamete activation. We demonstrate that PDI-Trans is a viable anti-malarial drug and vaccine target blocking malarial transmission with the use of PDI inhibitor bacitracin (98.21%/92.48% reduction in intensity/prevalence), and anti-PDI-Trans antibodies (66.22%/33.16% reduction in intensity/prevalence). To our knowledge, these results provide the first evidence that PDI function is essential for malarial transmission, and emphasize the potential of anti-PDI agents to act as anti-malarials, facilitating the future development of novel transmission-blocking interventions.

Background The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. Methods The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. Results A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. Conclusion A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

Vitellogenesis and oocyte maturation require anautogenous female Anopheles mosquitoes to obtain a bloodmeal from a vertebrate host. The bloodmeal is rich in proteins that are readily broken down into amino acids in the midgut lumen and absorbed by the midgut epithelial cells where they are converted into lipids and then transported to other tissues including ovaries. The stearoyl-CoA desaturase (SCD) plays a pivotal role in this process by converting saturated (SFAs) to unsaturated (UFAs) fatty acids; the latter being essential for maintaining cell membrane fluidity amongst other housekeeping functions. Here, we report the functional and phenotypic characterization of SCD1 in the malaria vector mosquito Anopheles coluzzii. We show that RNA interference (RNAi) silencing of SCD1 and administration of sterculic acid (SA), a small molecule inhibitor of SCD1, significantly impact on the survival and reproduction of female mosquitoes following blood feeding. Microscopic observations reveal that the mosquito thorax is quickly filled with blood, a phenomenon likely caused by the collapse of midgut epithelial cell membranes, and that epithelial cells are depleted of lipid droplets and oocytes fail to mature. Transcriptional profiling shows that genes involved in protein, lipid and carbohydrate metabolism and immunity-related genes are the most affected by SCD1 knock down (KD) in blood-fed mosquitoes. Metabolic profiling reveals that these mosquitoes exhibit increased amounts of saturated fatty acids and TCA cycle intermediates, highlighting the biochemical framework by which the SCD1 KD phenotype manifests as a result of a detrimental metabolic syndrome. Accumulation of SFAs is also the likely cause of the potent immune response observed in the absence of infection, which resembles an auto-inflammatory condition. These data provide insights into mosquito bloodmeal metabolism and lipid homeostasis and could inform efforts to develop novel interventions against mosquito-borne diseases., Author summary Female mosquitoes can become infected with malaria parasites upon ingestion of blood from an infected person and can transmit the disease when they bite another person some days later. The bloodmeal is rich in proteins which female mosquitoes use to develop their eggs after converting them first to saturated and then to unsaturated fatty acids inside their gut cells. Here, we present the characterization of the enzyme that mosquitoes use to convert saturated to unsaturated fatty acids and show that when this enzyme is eliminated or inhibited mosquitoes cannot produce eggs and die soon after they feed on blood. The mosquito death appears to be primarily associated with the collapse of their gut epithelial barrier due to the loss of cell membrane integrity, leading to their inner body cavity being filled with the ingested blood. These mosquitoes also suffer from an acute and detrimental auto-inflammatory condition due to mounting of a potent immune response in the absence of any infection. We conclude that this enzyme and the mechanism of converting blood-derived proteins to unsaturated fatty acids as a whole can be a good target of interventions aiming at limiting the mosquito abundance and blocking malaria transmission.

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

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

Malaria parasites develop and grow as oocysts in the mosquito for several days before being able to infect another human. During this time, mosquitoes take regular bloodmeals to replenish their nutrient and energy reserves needed for flight and reproduction. We hypothesized that supplemental bloodmeals are critical for oocyst growth and that experimental infection protocols, typically involving a single bloodmeal, cause nutritional stress to developing oocysts. Therefore, enumerating oocysts independently of their growth and differentiation state may lead to erroneous conclusions regarding the efficacy of malaria transmission blocking interventions. We tested this hypothesis in Anopheles coluzzii mosquitoes infected with human and rodent parasites Plasmodium falciparum and Plasmodium berghei, respectively. We find that oocyst growth rates decrease at late developmental stages as infection intensities increase; an effect exacerbated at very high infection intensities. Oocyst growth and differentiation can be restored by supplemental bloodmeals even at high infection intensities. We show that high infection intensities as well as starvation conditions reduce RNA Polymerase III activity in oocysts unless supplemental bloodmeals are provided. Our data suggest that oocysts respond to crowding and nutritional stress by employing a dormancy-like strategy and urge development of alternative methods to assess the efficacy of transmission blocking interventions.

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

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

Background Malaria vector mosquitoes acquire midgut microbiota primarily from their habitat. The homeostasis of these microbial communities plays an essential role in the mosquito longevity, the most essential factor in the mosquito vectorial capacity. Our recent study revealed that silencing genes involved in regulation of the midgut homeostasis including FN3D1, FN3D3 and GPRGr9 reduced the survival of female adult Anopheles arabiensis mosquitoes. In the present study, we investigate the stability of the gene silencing efficiency of mosquitoes reared in three different breeding conditions representing distinct larval habitat types: town brick pits in Jimma, flood pools in the rural land of Asendabo and roadside pools in Wolkite. Methods First-instar larvae of An. arabiensis mosquitoes were reared separately using water collected from the three breeding sites. The resulting adult females were micro-injected with dsRNA targeting the FN3D1 gene (AARA003032) and their survival was monitored. Control mosquitoes were injected with dsRNA Lacz. In addition, the load of midgut microbiota of these mosquitoes was determined using flow cytometry. Results Survival of naïve adult female mosquitoes differed between the three sites. Mosquitoes reared using water collected from brick pits and flood pools survived longer than mosquitoes reared using water collected from roadside. However, the FN3D1 gene silencing effect on survival did not differ between the three sites. Conclusions The present study revealed that the efficacy of FN3D1 gene silencing is not affected by variation in the larval habitat. Thus, silencing this gene has potential for application throughout sub-Saharan Africa.

Background Vector control remains the most important tool to prevent malaria transmission. However, it is now severely constrained by the appearance of physiological and behavioral insecticide resistance. Therefore, the development of new vector control tools is warranted. Such tools could include immunization of blood hosts of vector mosquitoes with mosquito proteins involved in midgut homeostasis (anti-mosquito vaccines) or genetic engineering of mosquitoes that can drive population-wide knockout of genes producing such proteins to reduce mosquito lifespan and malaria transmission probability. Methods To achieve this, candidate genes related to midgut homeostasis regulation need to be assessed for their effect on mosquito survival. Here, different such candidate genes were silenced through dsRNA injection in the naturally occurring Anopheles arabiensis mosquitoes and the effect on mosquito survival was evaluated. Results Significantly higher mortality rates were observed in the mosquitoes silenced for FN3D1 (AARA003032), FN3D3 (AARA007751) and GPRGr9 (AARA003963) genes as compared to the control group injected with dsRNA against a non-related bacterial gene (LacZ). This observed difference in mortality rate between the candidate genes and the control disappeared when gene-silenced mosquitoes were treated with antibiotic mixtures, suggesting that gut microbiota play a key role in the observed reduction of mosquito survival. Conclusions We demonstrated that interference with the expression of the FN3D1, FN3D3 or GPRGr9 genes causes a significant reduction of the longevity of An. arabiensis mosquito in the wild. Electronic supplementary material The online version of this article (10.1186/s13071-019-3414-y) contains supplementary material, which is available to authorized users.

International audience; Peptidoglycan recognition proteins (PGRPs) constitute the primary means of bacterial recognition in insects. Recent work in the model organism Drosophila has revealed the mechanisms by which the complement of PGRPs refine the sensitivity of different tissues to bacterial elicitors, permitting the persistence of commensal bacteria in the gut whilst maintaining vigilance against bacterial infection. Here, we use in vivo knockdowns and in vitro pull-down assays to investigate the role of the three major isoforms of the transmembrane receptor of the Imd pathway, PGRPLC, in basal immunity in the Anopheles coluzzii mosquito midgut. Our results indicate that the mosquito midgut is regionalized in its expression of immune effectors and of PGRPLC1. We show that PGRPLC1 and PGRPLC3 are pulled down with polymeric DAP-type peptidoglycan, while PGRPLC2 and PGRPLC3 co-precipitate in the presence of TCT, a peptidoglycan monomer. These data suggest that, as found in Drosophila, discrimination of polymeric and monomeric PGN by Anopheles PGRPLC participates in the regulation of the Imd pathway.

The complement-like pathway of the African malaria mosquito Anopheles gambiae provides protection against infection by diverse pathogens. A functional requirement for a core set of proteins during infections by rodent and human malaria parasites, bacteria, and fungi suggests a similar mechanism operates against different pathogens. However, the extent to which the molecular mechanisms are conserved is unknown. In this study we probed the biochemical responses of complement-like pathway to challenge by the Gram-positive bacterium Staphyloccocus aureus. Western blot analysis of the hemolymph revealed that S. aureus challenge activates a TEP1 convertase-like activity and promotes the depletion of the protein SPCLIP1. S. aureus challenge did not lead to an apparent change in the abundance of the LRIM1/APL1C complex compared to challenge by the Gram-negative bacterium, Escherichia coli. Following up on this observation using a panel of LRIM1 and APL1C antibodies, we found that E. coli challenge, but not S. aureus, specifically activates a protease that cleaves the C-terminus of APL1C. Inhibitor studies in vivo and in vitro protease assays suggest that a serine protease is responsible for APL1C cleavage. This study reveals that despite different challenges converging on activation of a TEP1 convertase-like activity, the mosquito complement-like pathway also includes pathogen-specific reactions.

Chikungunya is a viral disease transmitted to humans primarily via the bites of infected Aedes mosquitoes. The virus caused a major epidemic in the Indian Ocean in 2004, affecting millions of inhabitants, while cases have also been observed in Europe since 2007. We developed a stochastic spatiotemporal model of Aedes albopictus-borne chikungunya transmission based on our recently developed environmentally-driven vector population dynamics model. We designed an integrated modelling framework incorporating large-scale gridded climate datasets to investigate disease outbreaks on Reunion Island and in Italy. We performed Bayesian parameter inference on the surveillance data, and investigated the validity and applicability of the underlying biological assumptions. The model successfully represents the outbreak and measures of containment in Italy, suggesting wider applicability in Europe. In its current configuration, the model implies two different viral strains, thus two different outbreaks, for the two-stage Reunion Island epidemic. Characterisation of the posterior distributions indicates a possible relationship between the second larger outbreak on Reunion Island and the Italian outbreak. The model suggests that vector control measures, with different modes of operation, are most effective when applied in combination: adult vector intervention has a high impact but is short-lived, larval intervention has a low impact but is long-lasting, and quarantining infected territories, if applied strictly, is effective in preventing large epidemics. We present a novel approach in analysing chikungunya outbreaks globally using a single environmentally-driven mathematical model. Our study represents a significant step towards developing a globally applicable Ae. albopictus-borne chikungunya transmission model, and introduces a guideline for extending such models to other vector-borne diseases.

Clip domain serine protease homologs are widely distributed in insect genomes and play important roles in regulating insect immune responses, yet their exact functions remain poorly understood. Here, we show that CLIPA2, a clip domain serine protease homolog of Anopheles gambiae, regulates the consumption of the mosquito complement-like protein TEP1 during systemic bacterial infections. We provide evidence that CLIPA2 localizes to microbial surfaces in a TEP1-dependent manner whereby it negatively regulates the activity of a putative TEP1 convertase, which converts the full-length TEP1-F form into active TEP1cut. CLIPA2 silencing triggers an exacerbated TEP1-mediated response that significantly enhances mosquito resistance to infections with a broad class of microorganisms including Plasmodium berghei, Escherichia coli and the entomopathogenic fungus Beauveria bassiana. We also provide further evidence for the existence of a functional link between TEP1 and activation of hemolymph prophenoloxidase during systemic infections. Interestingly, the enhanced TEP1-mediated immune response in CLIPA2 knockdown mosquitoes correlated with a significant reduction in fecundity, corroborating the existence of a trade-off between immunity and reproduction. In sum, CLIPA2 is an integral regulatory component of the mosquito complement-like pathway which functions to prevent an overwhelming response by the host in response to systemic infections.

Arthropod borne diseases cause significant human morbidity and mortality and, therefore, efficient measures to control transmission of the disease agents would have great impact on human health. One strategy to achieve this goal is based on the manipulation of bacterial symbionts of vectors. Bacteria of the Gram-negative, acetic acid bacterium genus Asaia have been found to be stably associated with larvae and adults of the Southeast Asian malaria vector Anopheles stephensi, dominating the microbiota of the mosquito. We show here that after the infection of Anopheles gambiae larvae with Asaia the bacteria were stably associated with the mosquitoes, becoming part of the microflora of the midgut and remaining there for the duration of the life cycle. Moreover they were passed on to the next generation through vertical transmission. Additionally, we show that there is an increase in the developmental rate when additional bacteria are introduced into the organism which leads us to the conclusion that Asaia plays a yet undetermined crucial role during the larval stages. Our microarray analysis showed that the larval genes that are mostly affected are involved in cuticle formation, and include mainly members of the CPR gene family.

Aedes albopictus is a vector of dengue and chikungunya viruses in the field, along with around 24 additional arboviruses under laboratory conditions. As an invasive mosquito species, Ae. albopictus has been expanding in geographical range over the past 20 years, although the poleward extent of mosquito populations is limited by winter temperatures. Nonetheless, population densities depend on environmental conditions and since global climate change projections indicate increasing temperatures and altered patterns of rainfall, geographic distributions of previously tropical mosquito species may change. Although mathematical models can provide explanatory insight into observed patterns of disease prevalence in terms of epidemiological and entomological processes, understanding how environmental variables affect transmission is possible only with reliable model parameterisation, which, in turn, is obtained only through a thorough understanding of the relationship between mosquito biology and environmental variables. Thus, in order to assess the impact of climate change on mosquito population distribution and regions threatened by vector-borne disease, a detailed understanding (through a synthesis of current knowledge) of the relationship between climate, mosquito biology, and disease transmission is required, but this process has not yet been undertaken for Ae. albopictus. In this review, the impact of temperature, rainfall, and relative humidity on Ae. albopictus development and survival are considered. Existing Ae. albopictus populations across Europe are mapped with current climatic conditions, considering whether estimates of climatic cutoffs for Ae. albopictus are accurate, and suggesting that environmental thresholds must be calibrated according to the scale and resolution of climate model outputs and mosquito presence data.

The larval stages of malaria vector mosquitoes develop in water pools, feeding mostly on microorganisms and environmental detritus. Richness in the nutrient supply to larvae influences the development and metabolism of larvae and adults. Here, we investigated the effects of larval diet on the development, microbiota content and permissiveness to Plasmodium of Anopheles coluzzii. We tested three fish diets often used to rear mosquitoes in the laboratory, including two pelleted diets, Dr. Clarke’s Pool Pellets and Nishikoi Fish Pellets, and one flaked diet, Tetramin Fish-Flakes. Larvae grow and develop faster and produce bigger adults when feeding on both types of pellets compared with flakes. This correlates with a higher microbiota load in pellet-fed larvae, in agreement with the known positive effect of the microbiota on mosquito development. Larval diet also significantly influences the prevalence and intensity of Plasmodium berghei infection in adults, whereby Nishikoi Fish Pellets-fed larvae develop into adults that are highly permissive to parasites and survive longer after infection. This correlates with a lower amount of Enterobacteriaceae in the midgut microbiota. Together, our results shed light on the influence of larval feeding on mosquito development, microbiota and vector competence; they also provide useful data for mosquito rearing.

Background The Anopheles gambiae complex consists of species that vary greatly in their capacity to transmit malaria. The mosquito immune system has been identified as a key factor that can influence whether Plasmodium infection establishes within the mosquito vector. This study was designed to investigate the immune responses of An. coluzzii, An. arabiensis and An. quadriannulatus mosquitoes. The first two mosquito species are major vectors of malaria in sub-Saharan Africa, while the third is thought to be a non-vector. Methods All three mosquito species were reared in mixed cultures. Their capacity to eliminate P. berghei and regulate midgut bacteria was examined. Results Our results revealed large differences in mosquito resistance to P. berghei. In all three mosquito species, immune reactions involving the complement system were triggered when the number of parasites that mosquitoes were challenged with exceeded a certain level, i.e. immune tolerance threshold. This threshold was markedly lower in An. quadriannulatus compared to An. coluzzii and An. arabiensis. We also demonstrated that the level of immune tolerance to P. berghei infection in the haemolymph is inversely correlated with the level of immune tolerance to microbiota observed in the midgut lumen after a blood meal. The malaria non-vector mosquito species, An. quadriannulatus was shown to have a much higher level of tolerance to microbiota in the midgut than An. coluzzii. Conclusions We propose a model whereby an increased tolerance to microbiota in the mosquito midgut results in lower tolerance to Plasmodium infection. In this model, malaria non-vector mosquito species are expected to have increased immune resistance in the haemocoel, possibly due to complement priming by microbiota elicitors. We propose that this strategy is employed by the malaria non-vector mosquito, An. quadriannulatus, while An. coluzzii has reduced tolerance to bacterial infection in the midgut and consequently reduced immune resistance to Plasmodium infection at the haemocoel level. An in-depth understanding of the molecular mechanisms regulating immune tolerance versus resistance in different mosquito vectors of malaria could guide the design of new vector and disease control strategies. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2109-5) contains supplementary material, which is available to authorized users.

Background The combined effects of multiple density-dependent, regulatory processes may have an important impact on the growth and stability of a population. In a malaria model system, it has been shown that the progression of Plasmodium berghei through Anopheles stephensi and the survival of the mosquito both depend non-linearly on parasite density. These processes regulating the development of the malaria parasite within the mosquito may influence the success of transmission-blocking interventions (TBIs) currently under development. Methods An individual-based stochastic mathematical model is used to investigate the combined impact of these multiple regulatory processes and examine how TBIs, which target different parasite life-stages within the mosquito, may influence overall parasite transmission. Results The best parasite molecular targets will vary between different epidemiological settings. Interventions that reduce ookinete density beneath a threshold level are likely to have auxiliary benefits, as transmission would be further reduced by density-dependent processes that restrict sporogonic development at low parasite densities. TBIs which reduce parasite density but fail to clear the parasite could cause a modest increase in transmission by increasing the number of infectious bites made by a mosquito during its lifetime whilst failing to sufficiently reduce its infectivity. Interventions with a higher variance in efficacy will therefore tend to cause a greater reduction in overall transmission than a TBI with a more uniform effectiveness. Care should be taken when interpreting these results as parasite intensity values in natural parasite-vector combinations of human malaria are likely to be significantly lower than those in this model system. Conclusions A greater understanding of the development of the malaria parasite within the mosquito is required to fully evaluate the impact of TBIs. If parasite-induced vector mortality influenced the population dynamics of Plasmodium species infecting humans in malaria endemic regions, it would be important to quantify the variability and duration of TBI efficacy to ensure that community benefits of control measures are not overestimated.

Symbiotic bacteria can have important implications in the development and competence of disease vectors. In Anopheles mosquitoes, the composition of the midgut microbiota is largely influenced by the larval breeding site, but the exact factors shaping this composition are currently unknown. Here, we examined whether the proximity to urban areas and seasons have an impact on the midgut microbial community of the two major malaria vectors in Africa, An. coluzzii and An. gambiae. Larvae and pupae were collected from selected habitats in two districts of Ghana during the dry and rainy season periods. The midgut microbiota of adults that emerged from these collections was determined by 454-pyrosequencing of the 16S ribosomal DNA. We show that in both mosquito species, Shewanellaceae constituted on average of 54% and 73% of the midgut microbiota from each site in the dry and rainy season, respectively. Enterobacteriaceae was found in comparatively low abundance below 1% in 22/30 samples in the dry season, and in 25/38 samples in the rainy season. Our data indicate that seasonality and locality significantly affect both the diversity of microbiota and the relative abundance of bacterial families with a positive impact of dry season and peri-urban settings.

Peptidoglycan recognition proteins (PGRPs) form a family of immune regulators that is conserved from insects to mammals. In the malaria vector mosquito Anophelescoluzzii, the peptidoglycan receptor PGRPLC activates the immune-deficiency (Imd) pathway limiting both the microbiota load and Plasmodium infection. Here, we carried out an RNA interference screen to examine the role of all 7 Anopheles PGRPs in infections with Plasmodium berghei and P. falciparum. We show that, in addition to PGRPLC, PGRPLA and PGRPS2/PGRPS3 also participate in antiparasitic defenses, and that PGRPLB promotes mosquito permissiveness to P. falciparum. We also demonstrate that following a mosquito blood feeding, which promotes growth of the gut microbiota, PGRPLA and PGRPLB positively and negatively regulate the activation of the Imd pathway, respectively. Our data demonstrate that PGRPs are important regulators of the mosquito epithelial immunity and vector competence.

Background NeemAzal ® (NA) is a quantified extract from seed kernels of neem, Azadirachta indica A.Juss. (Meliaceae), with a wide spectrum of biological properties, classically ascribed to its limonoid content. NA contains several azadirachtins (A to L), azadirachtin A (AzaA) being its main constituent. AzaA has been shown to inhibit microgamete formation of the rodent malaria parasite Plasmodium berghei , and NA was found to completely inhibit the transmission of Plasmodium berghei to Anopheles stephensi mosquitoes when administered to gametocytemic mice at a corresponding AzaA dose of 50 mg/kg before exposure to mosquitoes. Purpose The present study was aimed at i) assessing the pharmacodynamics and duration of action of NA and AzaA against P. berghei exflagellation in systemic circulation in mice and ii) elucidating the transmission blocking activity (TBA) of the main NA constituents. Study design The NA and AzaA pharmacodynamics on exflagellation were assessed through ex vivo exflagellation assays, while TBA of NA constituents was evaluated through in vitro ookinete development assay. Methods Pharmacodynamics experiments: Peripheral blood from P. berghei infected BALB/c mice with circulating mature gametocytes, were treated i.p. with 50 mg/kg and 100 mg/kg pure AzaA and with NeemAzal ® (Trifolio-M GmbH) at the corresponding AzaA concentrations. The effect magnitude and duration of action of compounds was estimated by counting exflagellation centers, formed by microgametocytes in process of releasing flagellated gametes, at various time points after treatment in ex vivo exflagellation tests. Ookinete Development Assay: The direct effects of NeemAzal ® and AzaA on ookinete development were measured by fluorescence microscopy after incubation of gametocytemic blood with various concentrations of test substances in microplates for 24 h. Results The exflagellation tests revealed an half-life of NA anti-plasmodial compounds of up to 7 h at a NA dose corresponding to 100 mg/kg equivalent dose of AzaA. The ookinete development assay showed an increased activity of NA against early sporogonic stages compared to that of AzaA. The IC 50 value determined for NA was 6.8 µg/ml (CI 95 : 5.95–7.86), about half of the AzaA IC 50 (12.4 µg/ml; CI 95 : 11.0–14.04). Conclusion The stronger activity of NA, when compared to AzaA, could not be explained by an additive or synergistic effect by other azadirachtins (B, D and I) present in NA. In fact, the addition of these compounds at 50 µM concentration to AzaA did not evidence any decrease of the IC 50 against early sporogonic stages to that obtained with AzaA alone. It is likely that other non-limonoid compounds present in NA may contribute to AzaA activity and enhanced pharmacodynamics against exflagellation both in vitro and in vivo .

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

Leucine-rich repeat–containing proteins are central to host defense in plants and animals. We show that in the mosquito Anopheles gambiae , two such proteins that antagonize malaria parasite infections, LRIM1 and APL1C, circulate in the hemolymph as a high-molecular-weight complex held together by disulfide bridges. The complex interacts with the complement C3-like protein, TEP1, promoting its cleavage or stabilization and its subsequent localization on the surface of midgut-invading Plasmodium berghei parasites, targeting them for destruction. LRIM1 and APL1C are members of a protein family with orthologs in other disease vector mosquitoes and appear to be important effectors in innate mosquito defenses against human pathogens.

VectorBase (http://www.vectorbase.org) is an NIAID-funded Bioinformatic Resource Center focused on invertebrate vectors of human pathogens. VectorBase annotates and curates vector genomes providing a web accessible integrated resource for the research community. Currently, VectorBase contains genome information for three mosquito species: Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus, a body louse Pediculus humanus and a tick species Ixodes scapularis. Since our last report VectorBase has initiated a community annotation system, a microarray and gene expression repository and controlled vocabularies for anatomy and insecticide resistance. We have continued to develop both the software infrastructure and tools for interrogating the stored data.

The African mosquito Anopheles gambiae is the major vector of human malaria. We report a genome-wide survey of mosquito gene expression profiles clustered temporally into developmental programs and spatially into adult tissue-specific patterns. Global expression analysis shows that genes that belong to related functional categories or that encode the same or functionally linked protein domains are associated with characteristic developmental programs or tissue patterns. Comparative analysis of our data together with data published from Drosophila melanogaster reveal an overall strong and positive correlation of developmental expression between orthologous genes. The degree of correlation varies, depending on association of orthologs with certain developmental programs or functional groups. Interestingly, the similarity of gene expression is not correlated with the coding sequence similarity of orthologs, indicating that expression profiles and coding sequences evolve independently. In addition to providing a comprehensive view of temporal and spatial gene expression during the A. gambiae life cycle, this large-scale comparative transcriptomic analysis has detected important evolutionary features of insect transcriptomes.

Background The scientific interest to understand the function and structure of the microbiota associated with the midgut of mosquito disease vectors is increasing. The advancement of such a knowledge has encountered challenges and limitations associated with conventional culture-based and PCR techniques. Methods Flow cytometry (FCM) combined with various cell marking dyes have been successfully applied in the field of ecological microbiology to circumvent the above shortcomings. Here, we describe FCM technique coupled with live/dead differential staining dyes SYBR Green I (SGI) and Propidium Iodide (PI) to quantify and study other essential characteristics of the mosquito gut microbiota. Results A clear discrimination between cells and debris, as well as between live and dead cells was achieved when the midgut homogenate was subjected to staining with 5 × 103 dilution of the SGI and 30 μM concentration of the PI. Reproducibly, FCM event collections produced discrete populations including non-fluorescent cells, SYBR positive cells, PI fluorescing cells and cells that fluoresce both in SYBR and PI, all these cell populations representing, respectively, background noise, live bacterial, dead cells and inactive cells with partial permeability to PI. The FCM produced a strong linear relationship between cell counts and their corresponding dilution factors (R2 = 0.987), and the technique has a better precision compared to qRT-PCR. The FCM count of the microbiota reached a peak load at 18 h post-feeding and started declining at 24 h. The present FCM technique also successfully applied to quantify bacterial cells in fixed midgut samples that were homogenized in 4 % PFA. Conclusion The FCM technique described here offers enormous potential and possibilities of integration with advanced molecular biochemical techniques for the study of the microbiota community in disease vector mosquitoes.

Background: Mosquito infection with malaria parasites depends on complex interactions between the mosquito immune response, the parasite developmental program and the midgut microbiota. Simultaneous monitoring of the parasite and bacterial dynamics is important when studying these interactions. PCR based methods of genomic DNA (gDNA) have been widely used, but their inability to discriminate between live and dead cells compromises their application. The alternative method of quantification of mRNA mainly reports on cell activity rather than density. Method: Quantitative real-time (qrt) PCR in combination with Propidium Monoazide (PMA) treatment (PMA-qrtPCR) has been previously used for selectively enumerating viable microbial cells. PMA penetrates damaged cell membranes and intercalates in the DNA inhibiting its PCR amplification. Here, we tested the potential of PMA-qrtPCR to discriminate between and quantify live and dead Plasmodium berghei malarial parasites and commensal bacteria in the midgut of Anopheles coluzzii Coetzee & Wilkerson 2013 (formerly An. gambiae M-form). Results: By combining microscopic observations with reverse transcriptase PCR (RT-PCR) we reveal that, in addition to gDNA, mRNA from dead parasites also persists inside the mosquito midgut, therefore its quantification cannot accurately reflect live-only parasites at the time of monitoring. In contrast, pre-treating the samples with PMA selectively inhibited qrtPCR amplification of parasite gDNA, with about 15 cycles (Ct-value) difference between PMA-treated and control samples. The limit of detection corresponds to 10 Plasmodium ookinetes. Finally, we show that the PMA-qrtPCR method can be used to quantify bacteria that are present in the mosquito midgut. Conclusion: The PMA-qrtPCR is a suitable method for quantification of viable parasites and bacteria in the midgut of Anopheles mosquitoes. The method will be valuable when studying the molecular interactions between the mosquito, the malaria parasite and midgut microbiota.

Background Medicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernoniaamygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodiumberghei and to validate the findings on field isolates of Plasmodium falciparum. Methods Aqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V.amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P.falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926. Results Ver-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An.stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 μg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines. Conclusions Vernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0812-2) contains supplementary material, which is available to authorized users.

Arguably one of the most important effects of climate change is the potential impact on human health. While this is likely to take many forms, the implications for future transmission of vector-borne diseases (VBDs), given their ongoing contribution to global disease burden, are both extremely important and highly uncertain. In part, this is owing not only to data limitations and methodological challenges when integrating climate-driven VBD models and climate change projections, but also, perhaps most crucially, to the multitude of epidemiological, ecological and socio-economic factors that drive VBD transmission, and this complexity has generated considerable debate over the past 10–15 years. In this review, we seek to elucidate current knowledge around this topic, identify key themes and uncertainties, evaluate ongoing challenges and open research questions and, crucially, offer some solutions for the field. Although many of these challenges are ubiquitous across multiple VBDs, more specific issues also arise in different vector–pathogen systems.

Background Herbal remedies are widely used in many malaria endemic countries to treat patients, in particular in the absence of anti-malarial drugs and in some settings to prevent the disease. Herbal medicines may be specifically designed for prophylaxis and/or for blocking malaria transmission to benefit both, the individual consumer and the community at large. Neem represents a good candidate for this purpose due to its inhibitory effects on the parasite stages that cause the clinical manifestations of malaria and on those responsible for infection in the vector. Furthermore, neem secondary metabolites have been shown to interfere with various physiological processes in insect vectors. This study was undertaken to assess the impact of the standardised neem extract NeemAzal® on the fitness of the malaria vector Anopheles stephensi following repeated exposure to the product through consecutive blood meals on treated mice. Methods Batches of An. stephensi mosquitoes were offered 5 consecutive blood meals on female BALB/c mice treated with NeemAzal® at an azadirachtin A concentration of 60, 105 or 150 mg/kg. The blood feeding capacity was estimated by measuring the haematin content of the rectal fluid excreted by the mosquitoes during feeding. The number of eggs laid was estimated by image analysis and their hatchability assessed by direct observations. Results A dose and frequency dependent impact of NeemAzal® treatment on the mosquito feeding capacity, oviposition and egg hatchability was demonstrated. In the 150 mg/kg treatment group, the mosquito feeding capacity was reduced by 50% already at the second blood meal and by 50 to 80% in all treatment groups at the fifth blood meal. Consequently, a 50 – 65% reduction in the number of eggs laid per female mosquito was observed after the fifth blood meal in all treatment groups. Similarly, after the fifth treated blood meal exposure, hatchability was found to be reduced by 62% and 70% in the 105 and 150 mg/kg group respectively. Conclusions The findings of this study, taken together with the accumulated knowledge on neem open the challenging prospects of designing neem-based formulations as multi-target phytomedicines exhibiting preventive, parasite transmission-blocking as well as anti-vectorial properties.

Malaria reduction is most efficiently achieved by vector control whereby human populations at high risk of contracting and transmitting the disease are protected from mosquito bites. Here, we identify the presence of antibiotics in the blood of malaria-infected people as a new risk of increasing disease transmission. We show that antibiotics in ingested blood enhance the susceptibility of Anopheles gambiae mosquitoes to malaria infection by disturbing their gut microbiota. This effect is confirmed in a semi-natural setting by feeding mosquitoes with blood of children naturally infected with Plasmodium falciparum. Antibiotic exposure additionally increases mosquito survival and fecundity, which are known to augment vectorial capacity. These findings suggest that malaria transmission may be exacerbated in areas of high antibiotic usage, and that regions targeted by mass drug administration programs against communicable diseases may necessitate increased vector control., The gut microbiota of malaria-transmitting mosquitoes contributes to the insects’ resistance to the parasite. Here, Gendrin et al. show that antibiotics in ingested human blood alter the mosquito gut microbiota and increase the insect’s survival, fecundity and susceptibility to the parasites.

Climate change can influence the transmission of vector-borne diseases (VBDs) through altering the habitat suitability of insect vectors. Here we present global climate model simulations and evaluate the associated uncertainties in view of the main meteorological factors that may affect the distribution of the Asian tiger mosquito ( Aedes albopictus ), which can transmit pathogens that cause chikungunya, dengue fever, yellow fever and various encephalitides. Using a general circulation model at 50 km horizontal resolution to simulate mosquito survival variables including temperature, precipitation and relative humidity, we present both global and regional projections of the habitat suitability up to the middle of the twenty-first century. The model resolution of 50 km allows evaluation against previous projections for Europe and provides a basis for comparative analyses with other regions. Model uncertainties and performance are addressed in light of the recent CMIP5 ensemble climate model simulations for the RCP8.5 concentration pathway and using meteorological re-analysis data (ERA-Interim/ECMWF) for the recent past. Uncertainty ranges associated with the thresholds of meteorological variables that may affect the distribution of Ae. albopictus are diagnosed using fuzzy-logic methodology, notably to assess the influence of selected meteorological criteria and combinations of criteria that influence mosquito habitat suitability. From the climate projections for 2050, and adopting a habitat suitability index larger than 70%, we estimate that approximately 2.4 billion individuals in a land area of nearly 20 million km 2 will potentially be exposed to Ae. albopictus . The synthesis of fuzzy-logic based on mosquito biology and climate change analysis provides new insights into the regional and global spreading of VBDs to support disease control and policy making.

International audience; A microarray containing approximately 20 000 expressed sequence tags (ESTs; 11 760 unique EST clusters) from the malaria vector, Anopheles gambiae, was used to monitor differences in global gene expression in two insecticide resistant and one susceptible strains. Statistical analysis identified 77 ESTs that were differentially transcribed among the three strains. These include the cytochrome P450 CYP314A1, over-transcribed in the DDT resistant ZAN/U strain, and many genes that belong to families not usually associated with insecticide resistance, such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism. Short-term (6 and 10 h) effects of exposure of the pyrethroid resistant RSP strain to permethrin were also detected. Several genes belonging to enzyme families already implicated in insecticide or xenobiotic detoxification were induced, including the carboxylesterase COEAE2F gene and members of the UDP-glucuronosyl transferase and nitrilase families.

To determine if gene expression of An. gambiae is modulated in response to o'nyong-nyong virus (ONNV) infection, we utilized cDNA microarrays including about 20 000 cDNAs. Gene expression levels of ONNV-infected female mosquitoes were compared to that of the uninfected control females harvested at 14 days postinfection. In response to ONNV infection, expression levels of 18 genes were significantly modulated, being at least two-fold up- or down-regulated. Quantitative real-time PCR analysis (qRT-PCR) further substantiated the differential expression of six of these genes in response to ONNV infection. These genes have similarity to a putative heat shock protein 70, DAN4, agglutinin attachment subunit, elongation factor 1 alpha and ribosomal protein L35. One gene, with sequence similarity to mitochondrial ribosomal protein L7, was down-regulated in infected mosquitoes. The expression levels and annotation of the differentially expressed genes are discussed in the context of host/virus interaction including host translation/replication factors, and intracellular transport pathways.

We show that, in the malaria vector Anopheles gambiae , expression of Cecropin 1 is regulated by REL2, an NF-κB-like transcription factor orthologous to Drosophila Relish. Through alternative splicing, REL2 produces a full-length (REL2-F) and a shorter (REL2-S) protein isoform lacking the inhibitory ankyrin repeats and death domain. RNA interference experiments show that, in contrast to Drosophila Relish, which responds solely to Gram-negative bacteria, the Anopheles REL2-F and REL2-S isoforms are involved in defense against the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli bacteria, respectively. REL2-F also regulates the intensity of mosquito infection with the malaria parasite, Plasmodium berghei . The adaptor IMD shares the same activities as REL2-F. Microarray analysis identified 10 additional genes regulated by REL2, including CEC3 , GAM1 , and LRIM1 .

Summary Background: The malaria parasite Plasmodium must complete a complex developmental life cycle within Anopheles mosquitoes before it can be transmitted into the human host. One day after mosquito infection, motile ookinetes traverse the midgut epithelium and, after exiting to its basal site facing the hemolymph, develop into oocysts. Previously, we have identified hemolymph factors that can antagonize or promote parasite development. Results: We profiled on a genomic scale the transcriptional responses of the A. gambiae midgut to P. berghei and showed that more than 7% of the assessed mosquito transcriptome is differentially regulated during invasion. The profiles suggested that actin- and microtubule-cytoskeleton remodeling is a major response of the epithelium to ookinete penetration. Other responses encompass components of innate immunity, extracellular-matrix remodeling, and apoptosis. RNAi-dependent gene silencing identified both parasite antagonists and agonists among regulators of actin dynamics and revealed that actin polymerization is inhibitory to the invading parasite. Combined transcriptional and reverse-genetic analysis further identified an unexpected dual role of the lipid-trafficking machinery of the hemolymph for both parasite and mosquito-egg development. Conclusions: We conclude that the determinants of malaria-parasite development in Anopheles include components not only of systemic humoral immunity but also of intracellular, local epithelial reactions. These results provide novel mechanistic insights for understanding malaria transmission in the mosquito vector.

As the malaria burden persists in most parts of the developing world, the concept of implementation of new strategies such as the use of genetically modified mosquitoes to control the disease continues to gain support. In Africa, which suffers most from malaria, mosquito vector populations are spread almost throughout the entire continent, and the parasite reservoir is big and continuously increasing. Moreover, malaria is transmitted by many species of anophelines with specific seasonal and geographical patterns. Therefore, a well designed, evolutionarily robust and publicly accepted plan aiming at population reduction or replacement is required. The task is twofold: to engineer mosquitoes with a genetic trait that confers resistance to malaria or causes population suppression; and, to drive the new trait through field populations. This review examines these two issues, and describes the groundwork that has been done towards understanding of the complex relation between the parasite and its vector.

Though lacking adaptive immunity, insects possess a powerful innate immune system, a genome-encoded defence machinery used to confront infections. Studies in the fruit fly Drosophila melanogaster revealed a remarkable capacity of the innate immune system to differentiate between and subsequently respond to different bacteria and fungi. However, hematophagous compared to non-hematophagous insects encounter additional blood-borne infectious agents, such as parasites and viruses, during their lifetime. Anopheles mosquitoes become infected with the malaria parasite Plasmodium during feeding on infected human hosts and may then transmit the parasite to new hosts during subsequent bites. Whether Anopheles has developed mechanisms to confront these infections is the subject of this review. Initially, we review our current understanding of innate immune reactions and give an overview of the Anopheles immune system as revealed through comparative genomic analyses. Then, we examine and discuss the capacity of mosquitoes to recognize and respond to infections, especially to Plasmodium, and finally, we explore approaches to investigate and potentially utilize the vector immune competence to prevent pathogen transmission. Such approaches constitute a new challenge for insect immunity research, a challenge for global health.

In much of Africa, the mosquito Anopheles gambiae is the major vector of human malaria, a devastating infectious disease caused by Plasmodium parasites. Vector and parasite interact at multiple stages and locations, and the nature and effectiveness of this reciprocal interaction determines the success of transmission. Many of the interactions engage the mosquito's innate immunity, a primitive but very effective defense system. In some cases, the mosquito kills the parasite, thus blocking the transmission cycle. However, not all interactions are antagonistic; some represent immune evasion. The sequence of the A. gambiae genome revealed numerous potential components of the innate immune system, and it established that they evolve rapidly, as summarized in the present review. Their rapid evolution by gene family expansion diversification as well as the prevalence of haplotype alleles in the best-studied families may reflect selective adaptation of the immune system to the exigencies of multiple immune challenges in a variety of ecologic niches. As a follow-up to the comparative genomic analysis, the development of functional genomic methodologies has provided novel opportunities for understanding the immune system and the nature of its interactions with the parasite. In this context, identification of both Plasmodium antagonists and protectors in the mosquito represents a significant conceptual advance. In addition to providing fundamental understanding of primitive immune systems, studies of mosquito interactions with the parasite open unprecedented opportunities for novel interventions against malaria transmission. The generation of transgenic mosquitoes that resist malaria infection in the wild and the development of antimalarial 'smart sprays' capable of disrupting interactions that are protective of the parasite, or reinforcing others that are antagonistic, represent technical challenges but also immense opportunities for improvement of global health.

Malaria transmission depends on the competence of some Anopheles mosquitoes to sustain Plasmodium development (susceptibility). A genetically selected refractory strain of Anopheles gambiae blocks Plasmodium development, melanizing, and encapsulating the parasite in a reaction that begins with tyrosine oxidation, and involves three quantitative trait loci. Morphological and microarray mRNA expression analysis suggest that the refractory and susceptible strains have broad physiological differences, which are related to the production and detoxification of reactive oxygen species. Physiological studies corroborate that the refractory strain is in a chronic state of oxidative stress, which is exacerbated by blood feeding, resulting in increased steady-state levels of reactive oxygen species, which favor melanization of parasites as well as Sephadex beads.

Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency (“dual haplotypes”) in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster . Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.

The complex gene expression responses of Anopheles gambiae to microbial and malaria challenges, injury, and oxidative stress (in the mosquito and/or a cultured cell line) were surveyed by using cDNA microarrays constructed from an EST-clone collection. The expression profiles were broadly subdivided into induced and down-regulated gene clusters. Gram + and Gram − bacteria and microbial elicitors up-regulated a diverse set of genes, many belonging to the immunity class, and the response to malaria partially overlapped with this response. Oxidative stress activated a distinctive set of genes, mainly implicated in oxidoreductive processes. Injury up- and down-regulated gene clusters also were distinctive, prominently implicating glycolysis-related genes and citric acid cycle/oxidative phosphorylation/redox-mitochondrial functions, respectively. Cross-comparison of in vivo and in vitro responses indicated the existence of tightly coregulated gene groups that may correspond to gene pathways.

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.