Protein Kinase C-Dependent Signaling Controls the Midgut Epithelial Barrier to Malaria Parasite Infection in Anopheline Mosquitoes

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members – PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC − in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies.     

Transmission Blocking Immunity in the Malaria Non-Vector Mosquito Anopheles quadriannulatus Species A

Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa, Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vector mosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus is largely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodium berghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits and manifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequent stages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and the thioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completely abolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatus into a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of natural refractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to control transmission of the disease.     

Chromobacterium Csp_P Reduces Malaria and Dengue Infection in Vector Mosquitoes and Has Entomopathogenic and In Vitro Anti-pathogen Activities

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito''s vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies.     

A new role for an old antimicrobial: lysozyme c-1 can function to protect malaria parasites in Anopheles mosquitoes

Background

Plasmodium requires an obligatory life stage in its mosquito host. The parasites encounter a number of insults while journeying through this host and have developed mechanisms to avoid host defenses. Lysozymes are a family of important antimicrobial immune effectors produced by mosquitoes in response to microbial challenge.

Methodology/Principal Findings

A mosquito lysozyme was identified as a protective agonist for Plasmodium. Immunohistochemical analyses demonstrated that Anopheles gambiae lysozyme c-1 binds to oocysts of Plasmodium berghei and Plasmodium falciparum at 2 and 5 days after infection. Similar results were observed with Anopheles stephensi and P. falciparum, suggesting wide occurrence of this phenomenon across parasite and vector species. Lysozyme c-1 did not bind to cultured ookinetes nor did recombinant lysozyme c-1 affect ookinete viability. dsRNA-mediated silencing of LYSC-1 in Anopheles gambiae significantly reduced the intensity and the prevalence of Plasmodium berghei infection. We conclude that this host antibacterial protein directly interacts with and facilitates development of Plasmodium oocysts within the mosquito.

Conclusions/Significance

This work identifies mosquito lysozyme c-1 as a positive mediator of Plasmodium development as its reduction reduces parasite load in the mosquito host. These findings improve our understanding of parasite development and provide a novel target to interrupt parasite transmission to human hosts.     

Cover Picture

Dissected midgut of Anopheles gambiae mosquito infected with GFP‐fluorescent Plasmodium falciparum malaria oocysts     

Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae

Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca²⁺ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.     

Plasmodium chabaudi: Expression of active recombinant chabaupain-1 and localization studies in Anopheles sp.

Plasmodium cysteine proteases have been shown to be immunogenic and are being used as malaria potential serodiagnostic markers and vaccine targets. Genes encoding two Plasmodium chabaudi cysteine proteases chabaupain-1 (CP-1) and chabaupain-2 (CP-2) were identified and further expressed in Escherichia coli. Solubilisation of recombinant CP-1 and CP-2 was achieved by decreasing the temperature of induction.Anopheles gambiae tissues infected with Plasmodium were analyzed by Western blotting using the anti-CP-1 antibody showing that CP-1 is only present in the A. gambiae midguts being absent from other infected mosquito biological material. Anti-CP-1 anti-serum recognized a 30 kDa band in P. chabaudi, Plasmodium berghei and Plasmodium yoelii lysates but does not recognize the recombinant CP-2 extracts suggesting high antibody specificity.     

Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

Background

An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus.

Methods

Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin.

Results

The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed.

Conclusion

This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate molecular diagnostic tool for detecting malaria parasites in field mosquito populations.     

A low-cost microfluidic chip for rapid genotyping of malaria-transmitting mosquitoes

Background

Vector control is one of the most effective measures to prevent the transmission of malaria, a disease that causes over 600,000 deaths annually. Around 30–40 Anopheles mosquito species are natural vectors of malaria parasites. Some of these species cannot be morphologically distinguished, but have behavioral and ecological differences. Emblematic of this is the Anopheles gambiae species complex. The correct identification of vector species is fundamental to the development of control strategies and epidemiological studies of disease transmission.

Methodology/Principal Findings

An inexpensive, disposable, field-deployable, sample-to-answer, microfluidic chip was designed, constructed, and tested for rapid molecular identification of Anopheles gambiae and Anopheles arabiensis. The chip contains three isothermal amplification reactors. One test reactor operates with specific primers to amplify Anopheles gambiae DNA, another with specific primers for Anopheles arabiensis DNA, and the third serves as a negative control. A mosquito leg was crushed on an isolation membrane. Two discs, laden with mosquito tissue, were punched out of the membrane and inserted into the two test chambers. The isolated, disc-bound DNA served as a template in the amplification processes. The amplification products were detected with intercalating fluorescent dye that was excited with a blue light-emitting diode. The emitted light was observed by eye and recorded with a cell-phone camera. When the target consisted of Anopheles gambiae, the reactor containing primers specific to An. gambiae lit up while the other two reactors remained dark. When the target consisted of Anopheles arabiensis, the reactor containing primers specific to An. arabiensis lit up while the other two reactors remained dark.

Conclusions/Significance

The microfluidic chip provides a means to identify mosquito type through molecular analysis. It is suitable for field work, allowing one to track the geographical distribution of mosquito populations and community structure alterations due to environmental changes and malaria intervention measures.     

Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene

Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector–parasite interactions. Here, we developed a simple in vivo double-stranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and reproducible silencing of Defensin. Analysis of the knockdown phenotype revealed that this peptide is required for the mosquito antimicrobial defense against Gram-positive bacteria. In contrast, in mosquitoes infected by Plasmodium berghei, no loss of mosquito viability and no significant effect on the development and morphology of the parasite midgut stages were observed in the absence of Defensin. We conclude that this peptide is not a major antiparasitic factor in A. gambiae in vivo. Our results open new perspectives for the study of mosquito gene function in vivo and provide a basis for genome-scale systematic functional screens by targeted gene silencing.     

AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system

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

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

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

Climate influences on the cost-effectiveness of vector-based interventions against malaria in elimination scenarios

Despite the dependence of mosquito population dynamics on environmental conditions, the associated impact of climate and climate change on present and future malaria remains an area of ongoing debate and uncertainty. Here, we develop a novel integration of mosquito, transmission and economic modelling to assess whether the cost-effectiveness of indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) against Plasmodium falciparum transmission by Anopheles gambiae s.s. mosquitoes depends on climatic conditions in low endemicity scenarios. We find that although temperature and rainfall affect the cost-effectiveness of IRS and/or LLIN scale-up, whether this is sufficient to influence policy depends on local endemicity, existing interventions, host immune response to infection and the emergence rate of insecticide resistance. For the scenarios considered, IRS is found to be more cost-effective than LLINs for the same level of scale-up, and both are more cost-effective at lower mean precipitation and higher variability in precipitation and temperature. We also find that the dependence of peak transmission on mean temperature translates into optimal temperatures for vector-based intervention cost-effectiveness. Further cost-effectiveness analysis that accounts for country-specific epidemiological and environmental heterogeneities is required to assess optimal intervention scale-up for elimination and better understand future transmission trends under climate change.