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An obligatory step in the complex life cycle of the malaria parasite is sporogony, which occurs during the oocyst stage in adult female Anopheles mosquitoes. Sporogony is metabolically demanding, and successful oocyst maturation is dependent on host lipids. In insects, lipid energy reserves are mobilized by adipokinetic hormones (AKHs). We hypothesized that Plasmodium falciparum infection activates Anopheles gambiae AKH signaling and lipid mobilization. We profiled the expression patterns of AKH pathway genes and AgAkh1 peptide levels in An. gambiae during starvation, after blood feeding, and following infection and observed a significant time-dependent up-regulation of AKH pathway genes and peptide levels during infection. Depletion of AgAkh1 and AgAkhR by RNAi reduced salivary gland sporozoite production, while synthetic AgAkh1 peptide supplementation rescued sporozoite numbers. Inoculation of uninfected female mosquitoes with supernatant from P. falciparum-infected midguts activated AKH signaling. Clearly, identifying the parasite molecules mediating AKH signaling in P. falciparum sporogony is paramount., (© 2023. The Author(s).)

Background: Plasmodium ovale is a neglected malarial parasite that can form latent hypnozoites in the human liver. Over the last decade, molecular surveillance studies of non-falciparum malaria in Africa have highlighted that P. ovale is circulating below the radar, including areas where Plasmodium falciparum is in decline. To eliminate malaria where P. ovale is endemic, a better understanding of its epidemiology, asymptomatic carriage, and transmission biology is needed., Methods: We performed a pilot study on P. ovale transmission as part of an ongoing study of human-to-mosquito transmission of P. falciparum from asymptomatic carriers. To characterize the malaria asymptomatic reservoir, cross-sectional qPCR surveys were conducted in Bagamoyo, Tanzania, over three transmission seasons. Positive individuals were enrolled in transmission studies of P. falciparum using direct skin feeding assays (DFAs) with Anopheles gambiae s.s. (IFAKARA strain) mosquitoes. For a subset of participants who screened positive for P. ovale on the day of DFA, we incubated blood-fed mosquitoes for 14 days to assess sporozoite development., Results: Molecular surveillance of asymptomatic individuals revealed a P. ovale prevalence of 11% (300/2718), compared to 29% (780/2718) for P. falciparum. Prevalence for P. ovale was highest at the beginning of the long rainy season (15.5%, 128/826) in contrast to P. falciparum, which peaked later in both the long and short rainy seasons. Considering that these early-season P. ovale infections were low-density mono-infections (127/128), we speculate many were due to hypnozoite-induced relapse. Six of eight P. ovale-infected asymptomatic individuals who underwent DFAs successfully transmitted P. ovale parasites to A. gambiae., Conclusions: Plasmodium ovale is circulating at 4-15% prevalence among asymptomatic individuals in coastal Tanzania, largely invisible to field diagnostics. A different seasonal peak from co-endemic P. falciparum, the capacity to relapse, and efficient transmission to Anopheles vectors likely contribute to its persistence amid control efforts focused on P. falciparum., (© 2022. The Author(s).)

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Howard, Bender, Khare, López-Gutiérrez, Nyasembe, Weiss, Simecka, Hamerly, Mao and Dinglasan.)

Gravid female Anopheles gambiae mosquitoes identify suitable oviposition sites through a repertoire of cues, but the influence of allelochemicals, especially root phytochemicals in modulating this behavior and impacting subsequent progeny bionomics remains unexplored. We addressed these questions in the malaria vector Anopheles gambiae and its invasive host plant Parthenium hysterophorus. Using chemical analysis combined with laboratory behavioral assays, we demonstrate that a blend of terpenes, namely α-pinene, α-phellandrene, β-phellandrene, 3-carene and (E)-caryophyllene emitted from P. hysterophorus root exudate treated-water attracted gravid females. However, fewer eggs (55%) hatched in this treatment than in control water (66%). The sesquiterpene lactone parthenin, identified in both the natural aquatic habitat harboring P. hysterophorus and root exudate-treated water was found to be responsible for the ovicidal effect. Moreover, larvae exposed to parthenin developed 2 to 3 days earlier but survived 4 to 5 days longer as adults (median larval survival time = 9 days (all replicates);11 to 12 days as adults) than the non-exposed control (median larval survival time = 11 days (reps 1 & 2), 12 days (rep 3); 6 to 7 days as adults). These results improve our understanding of the risk and benefits of oviposition site selection by gravid An. gambiae females and the role root exudate allelochemicals could play on anopheline bionomics, with potential implications in malaria transmission., (© 2021. The Author(s).)

Background: Extracts of the invasive weed Parthenium hysterophorus (Asteraceae) have been shown to possess larvicidal activity against a wide range of disease vectors. However, the phytochemicals responsible for the larvicidal activity from this plant remain unidentified. Here, we isolated the major sesquiterpene lactone, parthenin (1) from the plant and synthesized two derivatives [ethylene glycol (2) and azide (3) derivatives] targeting the α,β-unsaturated carbonyl group, previously known to account for its biological activity such as toxicity towards cells and microorganism. All three compounds were screened for larvicidal activity against the African malaria vector Anopheles gambiae., Results: The larval mortality of ethylene glycol derivative (2) and 2α-azidocoronopilin (3) were approximately two-four-fold higher than that of parthenin (1) and neem oil with LC 50 values of 37 and 66 mg L -1 , respectively. Parthenin (1) and the positive control, neem oil, had comparable median lethal concentration (LC 50 ) values of 154 and 121 mg L -1 , respectively. In assays with binary combinations of the three compounds, larvicidal activity followed the order: parthenin (1) + 2α-azidocoronopilin (3) (LC 50 = 14 mg L -1 ) > parthenin (1) + ethylene glycol derivative (2) (LC 50 = 109 mg L -1 ), > blend of 2α-azidocoronopilin (3) and ethylene glycol derivative (2) (LC 50 = 200 mg L -1 )., Conclusion: Structural modification of parthenin (1) through addition of hydroxyl groups increases its larvicidal effects. These findings advance the use of structural modification approach in the development of lead chemical molecules for potential exploitation in larval source management., (© 2020 Society of Chemical Industry.)

Objective: A fundamental understanding of redox homeostasis in Anopheles gambiae midgut cells under different oxidative conditions is missing. Such knowledge can aid in the development of new malaria transmission-blocking strategies aimed at disrupting natural homeostatic processes in the mosquito during Plasmodium parasite uptake (i.e. blood feeding). The aim of this study was to understand how the An. gambiae midgut regulates oxidative stress to reactive oxygen species (ROS), especially to a potent ROS-inducer such as tert-Butyl hydroperoxide (tBHP)., Results: Initial studies using quantitative immunoblot indicated that the expression of the classical antioxidant protein An. gambiae thioredoxin-1 (AgTrx-1) remained unchanged across challenges with different concentrations of tBHP suggesting that additional mechanisms to regulate ROS may be involved. We therefore conducted a global proteomic survey, which revealed that An. gambiae midguts under low (50 μM) and high (200 μM) tBHP concentrations were enriched in proteins indicative of ribosomal/nucleolar stress. Ribosomal stress is an inherent cellular response to an imbalance in ribosomal proteins (RPs) due to cellular stress such as oxidative stress. Our data suggest that ribosomal/nucleolar stress is the primary cellular response in An. gambiae midguts under tBHP challenge. Considering these results, we discuss harnessing the ribosomal stress response as a potential malaria transmission-blocking strategy.

The evolution of drug resistance is a recurrent problem that has plagued efforts to treat and control malaria. Recent emergence of artemisinin resistance in Southeast Asia underscores the need to develop novel antimalarials and identify new targetable pathways in Plasmodium parasites. Transmission-blocking approaches, which typically target gametocytes in the host bloodstream or parasite stages in the mosquito gut, are recognized collectively as a strategy that when used in combination with antimalarials that target erythrocytic stages will not only cure malaria but will also prevent subsequent transmission. We tested four derivatives of (+)-usnic acid, a metabolite isolated from lichens, for transmission-blocking activity against Plasmodium falciparum using the standard membrane feeding assay. For two of the derivatives, BT37 and BT122, we observed a consistent dose-response relationship between concentration in the blood meal and oocyst intensity in the midgut. To explore their mechanism of action, we used the murine model Plasmodium berghei and found that both derivatives prevent ookinete maturation. Using fluorescence microscopy, we demonstrated that in the presence of each compound zygote vitality was severely affected, and those that did survive failed to elongate and mature into ookinetes. The observed phenotypes were similar to those described for mutants of specific kinases (NEK2/NEK4) and of inner membrane complex 1 (IMC1) proteins, which are all vital to the zygote-to-ookinete transition. We discuss the implications of our findings and our high-throughput screening approach to identifying next generation, transmission-blocking antimalarials based on the scaffolds of these (+)-usnic acid derivatives.

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Progress in malaria control has led to a significant reduction of the malaria burden. Interventions that interrupt transmission are now needed to achieve the elimination goal. Transmission-blocking vaccines (TBV) that aim to prevent mosquito infections represent promising tools and several vaccine candidates targeting different stages of the parasite's lifecycle are currently under development. A mosquito-midgut antigen, the anopheline alanyl aminopeptidase (AnAPN1) is one of the lead TBV candidates; antibodies against AnAPN1 prevent ookinete invasion. In this study, we explored the transmission dynamics of Plasmodium falciparum in mosquitoes fed with anti-AnAPN1 monoclonal antibodies (mAbs) vs. untreated controls, and investigated whether the parasite genetic content affects or is affected by antibody treatment. Exposure to anti-AnAPN1 mAbs was efficient at blocking parasite transmission and the effect was dose-dependent. Genetic analysis revealed a significant sib-mating within P. falciparum infra-populations infecting one host, as measured by the strong correlation between Wright's F IS and multiplicity of infection. Treatments also resulted in significant decrease in F IS as a by-product of drop in infra-population genetic diversity and concomitant increase of apparent panmictic genotyping proportions. Genetic differentiation analyses indicated that mosquitoes fed on a same donor randomly sampled blood-circulating gametocytes. We did not detect trace of selection, as the genetic differentiation between different donors did not decrease with increasing mAb concentration and was not significant between treatments for each gametocyte donor. Thus, there is apparently no specific genotype associated with the loss of diversity under mAb treatment. Finally, the anti-AnAPN1 mAbs were effective at reducing mosquito infection and a vaccine aiming at eliciting anti-AnAPN1 mAbs has a strong potential to decrease the burden of malaria in transmission-blocking interventions without any apparent selective pressure on the parasite population., (Copyright © 2016. Published by Elsevier B.V.)

Background: Malaria parasites are transmitted by Anopheles mosquitoes. Although several studies have identified mosquito midgut surface proteins that are putatively important for Plasmodium ookinete invasion, only a few have characterized these protein targets and demonstrated transmission-blocking activity. Molecular information about these proteins is essential for the development of transmission-blocking vaccines (TBV). The aim of the present study was to test three monoclonal antibodies (mAbs), A-140, A-78 and A-10, for their ability to recognize antigens and block oocyst infection of the midgut of Anopheles albimanus, a major malaria vector in Latin America., Method: Western-blot of mAbs on antigens from midgut brush border membrane vesicles was used to select antibodies. Three mAbs were tested by membrane feeding assays to evaluate their potential transmission-blocking activity against Plasmodium berghei. The cognate antigens recognized by mAbs with oocyst-reducing activity were determined by immunoprecipitation followed by liquid chromatography tandem mass spectrometry., Results: Only one mAb, A-140, significantly reduced oocyst infection intensity. Hence, its probable protein target in the Anopheles albimanus midgut was identified and characterized. It recognized three high-molecular mass proteins from a midgut brush border microvilli vesicle preparation. Chemical deglycosylation assays confirmed the peptide nature of the epitope recognized by mAb A-140. Immunoprecipitation followed by proteomic identification with tandem mass spectrometry revealed five proteins, presumably extracted together as a complex. Of these, AALB007909 had the highest mascot score and corresponds to a protein with a myosin head motor domain, indicating that the target of mAb A-140 is probably myosin located on the microvilli of the mosquito midgut., Conclusion: These results provide support for the participation of myosin in mosquito midgut invasion by Plasmodium ookinetes. The potential inclusion of this protein in the design of new multivalent vaccine strategies for blocking Plasmodium transmission is discussed.

Fucoconjugates are key mediators of protein-glycan interactions in prokaryotes and eukaryotes. As examples, N-glycans modified with the non-mammalian core α1,3-linked fucose have been detected in various organisms ranging from plants to insects and are immunogenic in mammals. The rabbit polyclonal antibody raised against plant horseradish peroxidase (anti-HRP) is able to recognize the α1,3-linked fucose epitope and is also known to specifically stain neural tissues in the fruit fly Drosophila melanogaster. In this study, we have detected and localized the anti-HRP cross-reactivity in another insect species, the malaria mosquito vector Anopheles gambiae. We were able to identify and structurally elucidate fucosylated N-glycans including core mono- and difucosylated structures (responsible for anti-HRP cross reactivity) as well as a Lewis-type antennal modification on mosquito anionic N-glycans by applying enzymatic and chemical treatments. The three mosquito fucosyltransferase open reading frames (FucT6, FucTA and FucTC) required for the in vivo biosynthesis of the fucosylated N-glycan epitopes were identified in the Anopheles gambiae genome, cloned and recombinantly expressed in Pichia pastoris. Using a robust MALDI-TOF MS approach, we characterised the activity of the three recombinant fucosyltransferases in vitro and demonstrate that they share similar enzymatic properties as compared to their homologues from D. melanogaster and Apis mellifera. Thus, not only do we confirm the neural reactivity of anti-HRP in a mosquito species, but also demonstrate enzymatic activity for all its α1,3- and α1,6-fucosyltransferase homologues, whose specificity matches the results of glycomic analyses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however, AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission have remained elusive. Here we present the 2.65-Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profiles of three monoclonal antibodies (mAbs) to AnAPN1, including mAb 4H5B7, which effectively blocks transmission of natural strains of Plasmodium falciparum. Using the AnAPN1 structure, we map the conformation-dependent 4H5B7 neoepitope to a previously uncharacterized region on domain 1 and further demonstrate that nonhuman-primate neoepitope-specific IgG also blocks parasite transmission. We discuss the prospect of a new biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV.

Novel strategies to directly thwart malaria transmission are needed to maintain the gains achieved by current control measures. Transmission-blocking interventions (TBIs), namely vaccines and drugs targeting parasite or mosquito molecules required for vector-stage parasite development, have been recognized as promising approaches for preventing malaria transmission. However, the number of TBI targets is limited and their degree of conservation among the major vector-parasite systems causing human disease is unclear. Therefore, discovery and characterization of novel proteins involved in vector-stage parasite development of Plasmodium falciparum and Plasmodium vivax is paramount. We mined the recent Anopheles gambiae midgut lipid raft proteome for putative mosquito-derived TBI targets and characterized a secreted glycoconjugate of unknown function, AgSGU. We analyzed molecular variation in this protein among a range of anopheline mosquitoes, determined its transcriptomic and proteomic profiles, and conducted both standard and direct membrane feeding assays with P. falciparum (lab/field) and P. vivax (field) in An. gambiae and Anopheles dirus. We observed that α-AgSGU antibodies significantly reduced midgut infection intensity for both lab and field isolates of P. falciparum in An. gambiae and An. dirus. However, no transmission-reducing effects were noted when comparable concentrations of antibodies were included in P. vivax-infected blood meals. Although antibodies against AgSGU exhibit transmission-reducing activity, the high antibody titer required for achieving 80% reduction in oocyst intensity precludes its consideration as a malaria mosquito-based TBI candidate. However, our results suggest that P. falciparum and P. vivax ookinetes use a different repertoire of midgut surface glycoproteins for invasion and that α-AgSGU antibodies, as well as antibodies to other mosquito-midgut microvillar surface proteins, may prove useful as tools for interrogating Plasmodium-mosquito interactions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Malaria transmission-blocking vaccines (TBVs) represent a promising approach for the elimination and eradication of this disease. AnAPN1 is a lead TBV candidate that targets a surface antigen on the midgut of the obligate vector of the Plasmodium parasite, the Anopheles mosquito. In this study, we demonstrated that antibodies targeting AnAPN1 block transmission of Plasmodium falciparum and Plasmodium vivax across distantly related anopheline species in countries to which malaria is endemic. Using a biochemical and immunological approach, we determined that the mechanism of action for this phenomenon stems from antibody recognition of a single protective epitope on AnAPN1, which we found to be immunogenic in murine and nonhuman primate models and highly conserved among anophelines. These data indicate that AnAPN1 meets the established target product profile for TBVs and suggest a potential key role for an AnAPN1-based panmalaria TBV in the effort to eradicate malaria.

Malaria transmission-blocking (T-B) interventions are essential for malaria elimination. Small molecules that inhibit the Plasmodium ookinete-to-oocyst transition in the midgut of Anopheles mosquitoes, thereby blocking sporogony, represent one approach to achieving this goal. Chondroitin sulfate glycosaminoglycans (CS-GAGs) on the Anopheles gambiae midgut surface are putative ligands for Plasmodium falciparum ookinetes. We hypothesized that our synthetic polysulfonated polymer, VS1, acting as a decoy molecular mimetic of midgut CS-GAGs confers malaria T-B activity. In our study, VS1 repeatedly reduced midgut oocyst development by as much as 99% (P<0.0001) in mosquitoes fed with P. falciparum and Plasmodium berghei. Through direct-binding assays, we observed that VS1 bound to two critical ookinete micronemal proteins, each containing at least one von Willebrand factor A (vWA) domain: (i) circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) and (ii) vWA domain-related protein (WARP). By immunofluorescence microscopy, we observed that VS1 stains permeabilized P. falciparum and P. berghei ookinetes but does not stain P. berghei CTRP knockouts or transgenic parasites lacking the vWA domains of CTRP while retaining the thrombospondin repeat region. We produced structural homology models of the first vWA domain of CTRP and identified, as expected, putative GAG-binding sites on CTRP that align closely with those predicted for the human vWA A1 domain and the Toxoplasma gondii MIC2 adhesin. Importantly, the models also identified patches of electropositive residues that may extend CTRP's GAG-binding motif and thus potentiate VS1 binding. Our molecule binds to a critical, conserved ookinete protein, CTRP, and exhibits potent malaria T-B activity. This study lays the framework for a high-throughput screen of existing libraries of safe compounds to identify those with potent T-B activity. We envision that such compounds when used as partner drugs with current antimalarial regimens and with RTS,S vaccine delivery could prevent the transmission of drug-resistant and vaccine-breakthrough strains.

Malaria morbidity and mortality caused by both Plasmodium falciparum and Plasmodium vivax extend well beyond the African continent, and although P. vivax causes between 80 and 300 million severe cases each year, vivax transmission remains poorly understood. Plasmodium parasites are transmitted by Anopheles mosquitoes, and the critical site of interaction between parasite and host is at the mosquito's luminal midgut brush border. Although the genome of the "model" African P. falciparum vector, Anopheles gambiae, has been sequenced, evolutionary divergence limits its utility as a reference across anophelines, especially non-sequenced P. vivax vectors such as Anopheles albimanus. Clearly, technologies and platforms that bridge this substantial scientific gap are required in order to provide public health scientists with key transcriptomic and proteomic information that could spur the development of novel interventions to combat this disease. To our knowledge, no approaches have been published that address this issue. To bolster our understanding of P. vivax-An. albimanus midgut interactions, we developed an integrated bioinformatic-hybrid RNA-Seq-LC-MS/MS approach involving An. albimanus transcriptome (15,764 contigs) and luminal midgut subproteome (9,445 proteins) assembly, which, when used with our custom Diptera protein database (685,078 sequences), facilitated a comparative proteomic analysis of the midgut brush borders of two important malaria vectors, An. gambiae and An. albimanus.

Background: Human Malaria is transmitted by mosquitoes of the genus Anopheles. Transmission is a complex phenomenon involving biological and environmental factors of humans, parasites and mosquitoes. Among more than 500 anopheline species, only a few species from different branches of the mosquito evolutionary tree transmit malaria, suggesting that their vectorial capacity has evolved independently. Anopheles albimanus (subgenus Nyssorhynchus) is an important malaria vector in the Americas. The divergence time between Anopheles gambiae, the main malaria vector in Africa, and the Neotropical vectors has been estimated to be 100 My. To better understand the biological basis of malaria transmission and to develop novel and effective means of vector control, there is a need to explore the mosquito biology beyond the An. gambiae complex., Results: We sequenced the transcriptome of the An. albimanus adult female. By combining Sanger, 454 and Illumina sequences from cDNA libraries derived from the midgut, cuticular fat body, dorsal vessel, salivary gland and whole body, we generated a single, high-quality assembly containing 16,669 transcripts, 92% of which mapped to the An. darlingi genome and covered 90% of the core eukaryotic genome. Bidirectional comparisons between the An. gambiae, An. darlingi and An. albimanus predicted proteomes allowed the identification of 3,772 putative orthologs. More than half of the transcripts had a match to proteins in other insect vectors and had an InterPro annotation. We identified several protein families that may be relevant to the study of Plasmodium-mosquito interaction. An open source transcript annotation browser called GDAV (Genome-Delinked Annotation Viewer) was developed to facilitate public access to the data generated by this and future transcriptome projects., Conclusions: We have explored the adult female transcriptome of one important New World malaria vector, An. albimanus. We identified protein-coding transcripts involved in biological processes that may be relevant to the Plasmodium lifecycle and can serve as the starting point for searching targets for novel control strategies. Our data increase the available genomic information regarding An. albimanus several hundred-fold, and will facilitate molecular research in medical entomology, evolutionary biology, genomics and proteomics of anopheline mosquito vectors. The data reported in this manuscript is accessible to the community via the VectorBase website (http://www.vectorbase.org/Other/AdditionalOrganisms/).

Introduction: In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes., Methods: WE COMPARED TWO COMMONLY USED MOSQUITO FEEDING ASSAY PROCEDURES: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum., Results: 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94-2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68-2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52-0.70)., Conclusions: Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.

Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission-blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that five of the characterized Anopheles TBV candidates, including the leading Anopheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectrometry. We identified 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identified in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion.

HS (heparan sulfate) has been shown to be an important mediator of Plasmodium sporozoite homing and invasion of the liver, but the role of this glycosaminoglycan in mosquito vector host-sporozoite interactions is unknown. We have biochemically characterized the function of AgOXT1 (Anopheles gambiae peptide-O-xylosyltransferase 1) and confirmed that AgOXT1 can modify peptides representing model HS and chondroitin sulfate proteoglycans in vitro. Moreover, we also demonstrated that the mosquito salivary gland basal lamina proteoglycans are modified by HS. We used RNA interference-mediated knockdown of HS biosynthesis in A. gambiae salivary glands to determine whether Plasmodium falciparum sporozoites that are released from mosquito midgut oocysts use salivary gland HS as a receptor for tissue invasion. Our results suggest that salivary gland basal lamina HS glycosaminoglycans only partially mediate midgut sporozoite invasion of this tissue, and that in the absence of HS, the presence of other surface co-receptors is sufficient to facilitate parasite entry.

Background: Ookinete is the form of the malaria parasite that invades the mosquito midgut epithelium to initiate sporogony. Differentiation of ingested gametocytes into ookinetes in the mosquito midgut lumen and subsequent interaction with the lumenal surface of the midgut epithelium in preparation for invasion is a complex and multi-stepped process. To facilitate the study of these events in detail it is necessary to produce sufficient numbers of pure, fully mature and functional ookinetes. However, production of even a small number of Plasmodium falciparum ookinetes in vitro has proven to be a daunting task. Consequently, over the past four decades our collective understanding of the biology of this parasite form remains sorely deficient. This article reports on investigations of five different ookinete media, in an effort to improve the in vitro transformation efficiency of P. falciparum gametocytes into mature ookinetes and their infectivity of the mosquito midgut., Methods: Five different ookinete media were evaluated for their ability to support the differentiation of gametocytes into gametes and further into mature stage V ookinetes. Moreover, infectivity of the in vitro-transformed ookinetes was evaluated by feeding them to vector mosquitoes and measuring their ability to traverse the midgut and form oocysts., Results: One of the five media (medium E) was clearly superior in that the cultured ookinetes produced the largest number of oocysts when fed to mosquitoes. Key components were additions of human serum, human red blood cell lysate and mosquito pupal extract, resulting in the production of larger numbers of ookinetes able to develop into oocysts when fed to mosquitoes., Conclusion: This simple and practical improvement over the prevailing methodology will facilitate the investigation of how this important human malaria parasite initiates its development in the mosquito and will contribute to the understanding of its transmission biology.

The scavenger receptor family comprises transmembrane proteins involved in the recognition of polyanionic ligands. Several studies have established that members of this family are involved both in immunity and in developmental processes. In Drosophila melanogaster, one of the best characterized scavenger receptors is Croquemort, which participates in the recognition of apoptotic cells in the embryo. Although comparative genomic studies have revealed the presence of four orthologs of this receptor in the malaria vector Anopheles gambiae, little is known about their function. We have investigated the expression pattern of the four Croquemort orthologs during the mosquito life cycle. Croquemort transcripts SCRBQ2 and SCRBQ4 are expressed at all the developmental stages, while expression of Croquemort transcripts SCRBQ1 and SCRBQ3 is more restricted. We have also investigated the expression of the four Croquemort orthologs in the different organs of the adult female. Croquemort transcript SCRBQ2 is highly expressed in the A. gambiae female midgut. SCRBQ2 midgut gene expression was up-regulated after a non-infected or a Plasmodium berghei-infected blood meal, compared to its expression in midguts of sugar-fed females. Interestingly, knockdown of SCRBQ2 expression by dsRNA injection resulted in a 62.5% inhibition of oocyst formation, suggesting that SCRBQ2 plays a role in Plasmodium-mosquito interactions.

Malaria is a devastating disease. For transmission to occur, Plasmodium, the causative agent of malaria, must complete a complex developmental cycle in its mosquito vector. Thus, the mosquito is a potential target for disease control. Plasmodium ookinetes, which develop within the mosquito midgut, must first cross the midgut's peritrophic matrix (PM), a thick extracellular sheath that completely surrounds the blood meal. The PM poses a partial, natural barrier against parasite invasion of the midgut and it is speculated that modifications to the PM may lead to a complete barrier to infection. However, such strategies require thorough characterization of the structure of the PM. Here, we describe for the first time, the complete PM proteome of the main malaria vector, Anopheles gambiae. Altogether, 209 proteins were identified by mass spectrometry. Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. Lastly, we provide a model for the putative interactions among the proteins identified in this study.

The idea of malaria transmission-blocking vaccines (TBVs) surfaced more than two decades ago. Since then, the research paradigm focused on developing TBVs that target surface antigens of parasite sexual stages. Only recently has an effort emerged that flipped this paradigm, targeting antigens of the parasite's obligate invertebrate vector, the Anopheles mosquito. Here, we review the current state of knowledge of mosquito-based TBVs and discuss the utility of this approach for future vaccine development.

Malaria transmission entails development of the Plasmodium parasite in its insect vector, the Anopheles mosquito. Parasite invasion of the mosquito midgut is the critical first step and involves adhesion to host epithelial cell ligands. Partial evidence suggests that midgut oligosaccharides are important ligands for parasite adhesion; however, the identity of these glycans remains unknown. We have identified a population of chondroitin glycosaminoglycans along the apical midgut microvilli of Anopheles gambiae and further demonstrated ookinete recognition of these glycans in vitro. By repressing the expression of the peptide-O-xylosyltransferase homolog of An. gambiae by means of RNA interference, we blocked glycosaminoglycan chain biosynthesis, diminished chondroitin sulfate levels in the adult midgut, and substantially inhibited parasite development. We provide evidence for the in vivo role of chondroitin sulfate proteoglycans in Plasmodium falciparum invasion of the midgut and insight into the molecular mechanisms mediating parasite-mosquito interactions.

Transmission blocking vaccines (TBV) against mosquito midgut carbohydrate epitopes is a promising approach to curbing the spread of malaria. However, carbohydrates as immunogens can be problematic. Via the malaria transmission blocking monoclonal antibody, MG96, we isolated dodecapeptide mimics of the conserved, nominal mosquito carbohydrate epitope from a peptide-display library. Two peptide clones, bearing a constrained, consensus motif competitively inhibited MG96 reactivity with its nominal midgut microvillar antigen. However, rabbit polyclonal antisera against these synthetic peptides recognized heterologous mosquito midgut carbohydrate and protein epitopes along the midgut basal lamina. Consequently, antisera did not block parasite development within the mosquito vector. Therefore, it is imperative that peptides not only need to be functional mimics but also complete mimotopes to effectively direct the vertebrate immune response towards the nominal, protective carbohydrate epitope on mosquito microvilli.

In spite of research efforts to develop vaccines against the causative agent of human malaria, Plasmodium falciparum, effective control remains elusive. The predominant vaccine strategy focuses on targeting parasite blood stages in the vertebrate host. An alternative approach has been the development of transmission-blocking vaccines (TBVs). TBVs target antigens on parasite sexual stages that persist within the insect vector, anopheline mosquitoes, or target mosquito midgut proteins that are presumed to mediate parasite development. By blocking parasite development within the insect vector, TBVs effectively disrupt transmission and the resultant cascade of secondary infections. Using a mosquito midgut-specific mouse monoclonal antibody (MG96), we have partially characterized membrane-bound midgut glycoproteins in Anopheles gambiae and Anopheles stephensi. These proteins are present on the microvilli of midgut epithelial cells in both blood-fed and unfed mosquitoes, suggesting that the expression of the protein is not induced as a result of blood feeding. MG96 exhibits a dose-dependent blocking effect against Plasmodium yoelii development in An. stephensi. We achieved 100% blocking of parasite development in the mosquito midgut. Preliminary deglycosylation assays indicate that the epitope recognized by MG96 is a complex oligosaccharide. Future investigation of the carbohydrate epitope as well as gene identification should provide valuable insight into the possible mechanisms of ookinete attachment and invasion of mosquito midgut epithelial cells.