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Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins which are channels facilitating the movement of water and other solutes across membranes. We identify a new aquaporin in malaria parasites and demonstrate that it is essential for disease transmission through mosquitoes. Disruption of AQP2 in the human parasitePlasmodium falciparumand the rodent parasitePlasmodium bergheiblocks sporozoite production inside oocysts established on mosquito midguts, preventing parasite infection of salivary glands and transmission to a new host.In vivoepitope tagging of AQP2 inP. berghei, combined with immunofluorescence assays, reveals that the protein is localized in previously uncharacterized organelles found in the cytoplasm of gametocytes, ookinetes and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endolysosomal system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely related to other intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature not found in any aquaporins studied to date. This in conjunction with the requirement of AQP2 for malaria transmission suggests that AQP2 may be a fruitful new target of novel antimalarial interventions.

SummaryMalaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, sexual reproduction and complex regulation of gene expression and genotype-phenotype associations in the mosquito have hampered the development of screens in this key part of the parasite lifecycle. We designed a genetic approach in the rodent parasitePlasmodium berghei, which in conjunction with barcode sequencing allowed us to overcome the fertilization roadblock and screen for gametocyte-expressed genes required for parasite infection of the mosquitoAnopheles coluzzii. The results confirmed previous findings, validating our approach for scaling up, and identified new genes required for ookinete motility and mosquito midgut infection and for sporozoite development and oocyst egress and salivary gland infection. Our findings can assist efforts to study malaria transmission biology and develop new interventions to control disease transmission.

Background Plasmodium interspersed repeat (pir) is the largest multigene family in the genomes of most Plasmodium species. A variety of functions for the PIR proteins which they encode have been proposed, including antigenic variation, immune evasion, sequestration and rosetting. However, direct evidence for these is lacking. The repetitive nature of the family has made it difficult to determine function experimentally. However, there has been some success in using gene expression studies to suggest roles for some members in virulence and chronic infection. Methods Here pir gene expression was examined across the life cycle of Plasmodium berghei using publicly available RNAseq data-sets, and at high resolution in the intraerythrocytic development cycle using new data from Plasmodium chabaudi. Results Expression of pir genes is greatest in stages of the parasite which invade and reside in red blood cells. The marked exception is that liver merozoites and male gametocytes produce a very large number of pir gene transcripts, notably compared to female gametocytes, which produce relatively few. Within the asexual blood stages different subfamilies peak at different times, suggesting further functional distinctions. Representing a subfamily of its own, the highly conserved ancestral pir gene warrants further investigation due to its potential tractability for functional investigation. It is highly transcribed in multiple life cycle stages and across most studied Plasmodium species and thus is likely to play an important role in parasite biology. Conclusions The identification of distinct expression patterns for different pir genes and subfamilies is likely to provide a basis for the design of future experiments to uncover their function.

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.

Genetically modified (GM) mosquitoes expressing anti-plasmodial effectors propagating through wild mosquito populations by means of gene drive is a promising tool to support current malaria control strategies. The process of generating GM mosquitoes involves genetic transformation of mosquitoes from a laboratory colony and, often, interbreeding with other GM lines to cross in auxiliary traits. These mosquito colonies and GM lines thus often have different genetic backgrounds and GM lines are invariably highly inbred, which in conjunction with their independent rearing in the laboratory may translate to differences in their susceptibility to malaria parasite infection and life history traits. Here, we show that laboratory Anopheles gambiae colonies and GM lines expressing Cas9 and Cre recombinase vary greatly in their susceptibility to Plasmodium falciparum NF54 infection. Therefore, the choice of mosquitoes to be used as a reference when conducting infection or life history trait assays requires careful consideration. To address these issues, we established an experimental pipeline involving genetic crosses and genotyping of mosquitoes reared in shared containers throughout their lifecycle. We used this protocol to examine whether GM lines expressing the antimicrobial peptide (AMP) Scorpine in the mosquito midgut interfere with parasite infection and mosquito survival. We demonstrate that Scorpine expression in the Peritrophin 1 (Aper1) genomic locus reduces both P. falciparum sporozoite prevalence and mosquito lifespan; both these phenotypes are likely to be associated with the disturbance of the midgut microbiota homeostasis. These data lead us to conclude that the Aper1-Sco GM line could be used in proof-of-concept experiments aimed at mosquito population replacement, although the impact of its reduced fitness on the spread of the transgene through wild populations requires further investigation.

The Eastern Mediterranean and Middle East (EMME) region has rapid population growth, large differences in socio-economic levels between developed and developing countries, migration, increased water demand, and ecosystems degradation. The region is experiencing a significant warming trend with longer and warmer summers, increased frequency and severity of heat waves, and a drier climate. While climate change plays an important role in contributing to political instability in the region through displacement of people, food insecurity, and increased violence, it also increases the risks of vector-, water-, and food-borne diseases. Poorer and less educated people, young children and the elderly, migrants, and those with long-term health problems are at highest risk. A result of the inequalities among EMME countries is an inconsistency in the availability of reliable evidence about the impacts on infectious diseases. To help address this gap, a search of the literature was conducted as a basis for related recommended responses and suggested actions for preparedness and prevention. Since climate change already impacts the health of vulnerable populations in the EMME and will have a greater impact in future years, risk assessment and timely design and implementation of health preparedness and adaptation strategies are essential. Joint national and cross-border infectious diseases management systems for more effective preparedness and prevention are needed, supported by interventions that improve the environment. Without such cooperation and effective interventions, climate change will lead to an increasing morbidity and mortality in the EMME from infectious diseases, with a higher risk for the most vulnerable populations.

Genetically modified (GM) mosquitoes expressing anti-plasmodial effectors propagating through wild mosquito populations by means of gene drive is a promising tool to support current malaria control strategies. The process of generating GM mosquitoes involves genetic transformation of mosquitoes from a laboratory colony and, often, interbreeding with other GM lines to cross in auxiliary traits. These mosquito colonies and GM lines thus often have different genetic backgrounds and GM lines are invariably highly inbred, which in conjunction with their independent rearing in the laboratory may translate to differences in their susceptibility to malaria parasite infection and life history traits. Here, we show that laboratory Anopheles gambiae colonies and GM lines expressing Cas9 and Cre recombinase vary greatly in their susceptibility to Plasmodium falciparum NF54 infection. Therefore, the choice of mosquitoes to be used as a reference when conducting infection or life history trait assays requires careful consideration. To address these issues, we established an experimental pipeline involving genetic crosses and genotyping of mosquitoes reared in shared containers throughout their lifecycle. We used this protocol to examine whether GM lines expressing the antimicrobial peptide (AMP) Scorpine in the mosquito midgut interfere with parasite infection and mosquito survival. We demonstrate that Scorpine expression in the Peritrophin 1 (Aper1) genomic locus reduces both P. falciparum sporozoite prevalence and mosquito lifespan; both these phenotypes are likely to be associated with the disturbance of the midgut microbiota homeostasis. These data lead us to conclude that the Aper1-Sco GM line could be used in proof-of-concept experiments aimed at mosquito population replacement, although the impact of its reduced fitness on the spread of the transgene through wild populations requires further investigation.

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.

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.

The cosmopolitan insect-pathogenic fungus and popular biocontrol agent Beauveria bassiana can be used to control Anopheles mosquito populations and restrict the spread of malaria, the deadliest vector-borne infectious disease in the world caused by the protozoan parasite Plasmodium. Here, we establish that infection with a double-stranded (ds)RNA mycovirus, Beauveria bassiana polymycovirus (BbPmV)-1, significantly reduces B. bassiana virulence against A. coluzzii, the main vector of malaria. The BbPmV-1-mediated hypovirulence can be at least partially attributed to slow fungal growth on the mosquitos. Analysis of the dual next-generation sequencing of the B. bassiana and A. coluzzii transcriptomes provided insight into the molecular mechanisms of the BbPmV-1-mediated effects. BbPmV-1-free B. bassiana has a wide impact on the A. coluzzii transcriptome, affecting immunity and metabolism, and led to the identification of novel immune response proteins. BbPmV-1 regulates the gene expression profile of its fungal host, directing the use of available resources towards sporulation and suppressing the mosquito immune system. Additionally, BbPmV-1-infected and -free B. bassiana strains differentially modulate mosquito gut microbiota; the former reduces the bacterial genus Elizabethkingia and the latter Serratia. Co-transfection of mosquitos with B. bassiana and P. berghei revealed a reduction of ookinetes in the presence of BbPmV-1, potentially due to the upregulation of a mycotoxin. Finally, BbPmV-1-mediated hypovirulence is at least partially dependent on the A. coluzzii RNAi pathway, and silencing of the dicer-2 gene restores virulence. Taken together, our data clearly demonstrate the crucial role of mycovirus infection in mediating B. bassiana virulence against A. coluzzii and suggest that BbPmV-1 protects A. coluzzii from B. bassiana, the mosquito’s own immune system, potentially harmful gut microbiota, and Plasmodium parasites.

The mosquito microbiota reduces the vector competence of Anopheles to Plasmodium and affects host fitness, it is therefore considered as a potential target to reduce malaria transmission. While immune induction, secretion of antimicrobials and metabolic competition are three typical mechanisms of microbiota-mediated protection against invasive pathogens in mammals, the involvement of metabolic competition or mutualism in mosquito-microbiota and microbiota-Plasmodium interactions has not been investigated. Here, we describe a metabolome analysis of the midgut of Anopheles coluzzii provided with a sugar-meal or a non-infectious blood-meal, under conventional or antibiotic-treated conditions. We observed that the antibiotic treatment affects the tricarboxylic acid cycle and nitrogen metabolism, notably resulting in decreased abundance of free amino acids. Linking our results with published data, we identified pathways which may participate in microbiota-Plasmodium interactions via metabolic interactions or immune modulation and thus would be interesting candidates for future functional studies.

The mosquito microbiota reduces the vector competence of Anopheles to Plasmodium and affects host fitness, it is therefore considered as a potential target to reduce malaria transmission. While immune induction, secretion of antimicrobials and metabolic competition are three typical mechanisms of microbiota-mediated protection against invasive pathogens in mammals, the involvement of metabolic competition or mutualism in mosquito-microbiota and microbiota-Plasmodium interactions has not been investigated. Here, we describe a metabolome analysis of the midgut of An. coluzzii provided with a sugar-meal or a blood-meal, under conventional or antibiotic-treated conditions. We observed that the antibiotic treatment affects the tricarboxylic acid cycle and nitrogen metabolism, notably resulting in decreased abundance of free amino acids. Linking our results with published data, we identified candidate pathways which may participate in microbiota/Plasmodium interactions via metabolic interactions or immune modulation.

The mosquito microbiota reduces the vector competence of Anopheles to Plasmodium and affects host fitness; it is therefore considered as a potential target to reduce malaria transmission. While immune induction, secretion of antimicrobials and metabolic competition are three typical mechanisms of microbiota-mediated protection against invasive pathogens in mammals, the involvement of metabolic competition or mutualism in mosquito-microbiota and microbiota-Plasmodium interactions has not been investigated. Here, we describe a metabolome analysis of the midgut of Anopheles coluzzii provided with a sugar-meal or a non-infectious blood-meal, under conventional or antibiotic-treated conditions. We observed that the antibiotic treatment affects the tricarboxylic acid cycle and nitrogen metabolism, notably resulting in decreased abundance of free amino acids. Linking our results with published data, we identified pathways which may participate in microbiota-Plasmodium interactions via metabolic interactions or immune modulation and thus would be interesting candidates for future functional studies.

Vitellogenesis and oocyte maturation require anautogenous femaleAnophelesmosquitoes 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 mosquitoAnopheles coluzzii. We show that RNA interference (RNAi) silencing ofSCD1and 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 bySCD1knock 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 theSCD1KD 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.

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 accepted testing pathway of gene drives for malaria eradication.

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.

Malaria transmission requires Plasmodium parasites to successfully infect a female Anopheles mosquito, surviving a series of robust innate immune responses. Understanding how parasites evade these responses can highlight new ways to block malaria transmission. We show that ookinete and sporozoite surface protein PIMMS43 is required for Plasmodium ookinete evasion of the Anopheles coluzzii complement-like system and for sporogonic development in the oocyst. Disruption of P. berghei PIMMS43 triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing the complement-like system restores ookinete-to-oocyst transition. 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 P. falciparum populations indicates allelic adaptation to sympatric vector populations. These data significantly add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of interventions aiming at malaria transmission blocking.Author summaryMalaria is a devastating disease transmitted among humans through mosquito bites. Mosquito control has significantly reduced clinical malaria cases and deaths in the last decades. However, as mosquito resistance to insecticides is becoming widespread impacting on current control tools, such as insecticide impregnated bed nets and indoor spraying, new interventions are urgently needed, especially those that target disease transmission. Here, we characterize a protein found on the surface of malaria parasites, which serves to evade the mosquito immune system ensuring disease transmission. Neutralization of PIMMS43, either by eliminating it from the parasite genome or by pre-incubating parasites with antibodies that bind to the protein, is shown to inhibit mosquito infection by malaria parasites. Differences in PIMMS43 detected between malaria parasite populations sampled across Africa suggest that these populations have adapted for transmission by different mosquito vectors that are also differentially distributed across the continent. We conclude that interventions targeting PIMMS43 could block malaria parasites inside mosquitoes before they can infect humans.

Epigenetic regulation of gene expression is an important attribute in the survival and adaptation of the malaria parasite Plasmodium in its human host. Our understanding of epigenetic regulation of gene expression in Plasmodium developmental stages beyond asexual replication in the mammalian host is sparse. We used chromatin immune-precipitation (ChIP) and RNA sequencing to create an epigenetic and transcriptomic map of the murine parasite Plasmodium berghei development from asexual blood stages to male and female gametocytes, and finally, to ookinetes. We show that heterochromatin 1 (HP1) almost exclusively associates with variantly expressed gene families at subtelomeric regions and remains stable across stages and various parasite lines. Variant expression based on heterochromatic silencing is observed only in very few genes. In contrast, the active histone mark histone 3 Lysine 9 acetylation (H3K9ac) is found between heterochromatin boundaries and occurs as a sharp peak around the start codon for ribosomal protein genes. H3K9ac occupancy positively correlates with gene transcripts in asexual blood stages, male gametocytes and ookinetes. Interestingly, H3K9ac occupancy does not correlate with transcript abundance in female gametocytes. Finally, we identify novel DNA motifs upstream of ookinete-specific genes thought to be involved in transcriptional activation upon fertilization.

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.

SummaryInhibiting 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. The 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 the PDI inhibitor bacitracin (98.21%/92.48% reduction in intensity/prevalence), and anti-PDI-Trans peptide antibodies (66.22%/33.16% reduction in intensity/prevalence). To our knowledge, these results provide the first primary evidence that protein disulphide isomerase 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 compounds or vaccines.

BackgroundIn the fight against malaria, vector control remains the most important tool, butit is now severely constrained by the spread of insecticide or behavioral resistance by mosquito populations. Therefore, new vector control tools are warranted. Such novel tools include anti-mosquito vaccines or mosquito genetic modifications targeting the mosquito midgut homeostasis and reducing the mosquito lifespan beyond a stage they can transmit malaria.MethodsWe assessed the effect of RNA interference silencing of the midgut homeostasis regulators FN3D1, FN3D2, FN3D3, GPRGR9 and PGRPLC3 in populations of Anopheles arabiensis reared at nearly natural setting. We monitored the survival of gene-silenced mosquitoes and assessed the load of their midgut microbiota using flow cytometry. The effect of gene silencing was modeled by the Cox proportional hazards frailty model, and bacterial counts were first log transformed and then compared by a mixed model.ResultSignificantly higher mortality rates were observed for the FN3D1 (Hazard ratio =1.64, P=0.004), FN3D3 (HR=1.79, PGPRGr9 silenced mosquitoes (HR=2.00, PFN3D1 equal to 2.66 (95%CI: [0.94;7.57]) but no statistically significant difference could be demonstrated. Interestingly, there was a strong correlation (r=0.61) between the mortality hazard ratio and the bacterial count ratio of the gene-silenced mosquitoes. Increased mortality rates were reversed when the gene-silenced mosquitoes were treated with antibiotic mixtures suggesting that gut microbiota play a key role in the observed reduction of mosquito survival.ConclusionWe demonstrate that interfering with the expression of theFN3D1, FN3D3 or GPRGr9 genes can cause a significant reduction of the longevity of An. arabiensis mosquitoes due to the disruption of the mosquito gut homeostasis.

First generation CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. A number of challenges for their use in the area-wide genetic control of vector-borne disease have been identified. These include the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and the practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. To address these challenges, we have evaluated the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including both the drive and the anti-pathogen effector elements directly embedded within endogenous genes – an arrangement which we refer to as gene “hijacking”. This design would allow autonomous and non-autonomous IGD traits and strains to be generated, tested, optimized, regulated and imported independently. We performed quantitative modelling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population while hedging drive over multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target-site variation. IGD thus has the potential to yield more durable and flexible population replacement traits.

A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes – an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy., Summary: Here, we describe and model a novel gene drive strategy – integral gene drive – for population replacement based on the hijacking of endogenous genes.

Summary The malaria parasite develops sexually in the mosquito midgut upon entry with the ingested blood meal before it can invade the midgut epithelium and embark on sporogony. Recent data have identified a number of distinct transcriptional programmes operating during this critical phase of the parasite life cycle. We aimed at characterizing the parental contribution to these transcriptional programmes and establish the genetic framework that would guide further studies of P lasmodium zygotic development and ookinete‐to‐oocyst transition. To achieve this we used in vitro and in vivo cross‐fertilization experiments of various parasite lines expressing fluorescent reporters under the control of constitutive and stage‐specific promoters. The results revealed that the zygote/ookinete stage exhibits a maternal phenotype with respect to constitutively expressed reporters, which is derived from either maternal mRNA inheritance or transcription of the maternal allele. The respective paternal alleles are silenced in the zygote/ookinete but reactivated after midgut invasion and transformation to oocyst. Transcripts specifically produced in the zygote/ookinete are synthesized de novo by both parental alleles. These findings highlight a putative role of epigenetic regulation of P lasmodium zygotic development and add substantially to the emerging picture of the molecular mechanisms regulating this important stage of malaria transmission.