Novel factors of Anopheles gambiae haemocyte immune response to Plasmodium berghei infection

Background Insect haemocytes mediate cellular immune responses (e.g., phagocytosis) and contribute to the synthesis of humoral immune factors. In previous work, a genome-wide molecular characterization of Anopheles gambiae circulating haemocytes was followed by functional gene characterization using cell-based RNAi screens. Assays were carried out to investigate the role of selected haemocyte-specific or enriched genes in phagocytosis of bacterial bio-particles, expression of the antimicrobial peptide cecropin1, and basal and induced expression of the mosquito complement factor LRIM1 (leucine-rich repeat immune gene I). Findings Here we studied the impact of a subset of genes (37 candidates) from the haemocyte-specific dsRNA collection on the development of Plasmodium in the mosquito by in vivo gene silencing. Our screening identifies 10 novel factors with a role in the mosquito response to Plasmodium. Analysis of in vivo screening phenotypes reveals a significant anti-correlation between the prevalence of oocysts and melanised ookinetes. Conclusions Among novel immune genes are putative pattern recognition proteins (leucine-rich repeat, fibrinogen-domain and R-type lectins), immune modulation and signalling proteins (LPS-induced tumor necrosis factor alpha factor, LITAF and CLIP proteases), and components of extracellular matrix such as laminin and collagen. Additional identified proteins such as the storage protein hexamerin and vesicular-type ATPase (V-ATPase) are associated for the first time with the mosquito response against Plasmodium

Sequence-structure-function relations of the mosquito leucine-rich repeat immune proteins.

<p>Abstract</p> <p>Background</p> <p>The discovery and characterisation of factors governing innate immune responses in insects has driven the elucidation of many immune system components in mammals and other organisms. Focusing on the immune system responses of the malaria mosquito, <it>Anopheles gambiae</it>, has uncovered an array of components and mechanisms involved in defence against pathogen infections. Two of these immune factors are LRIM1 and APL1C, which are leucine-rich repeat (LRR) containing proteins that activate complement-like defence responses against malaria parasites. In addition to their LRR domains, these leucine-rich repeat immune (LRIM) proteins share several structural features including signal peptides, patterns of cysteine residues, and coiled-coil domains.</p> <p>Results</p> <p>The identification and characterisation of genes related to <it>LRIM1 </it>and <it>APL1C </it>revealed putatively novel innate immune factors and furthered the understanding of their likely molecular functions. Genomic scans using the shared features of <it>LRIM1 </it>and <it>APL1C </it>identified more than 20 <it>LRIM</it>-like genes exhibiting all or most of their sequence features in each of three disease-vector mosquitoes with sequenced genomes: <it>An. gambiae</it>, <it>Aedes aegypti</it>, and <it>Culex quinquefasciatus</it>. Comparative sequence analyses revealed that this family of mosquito <it>LRIM</it>-like genes is characterised by a variable number of 6 to 14 LRRs of different lengths. The "Long" LRIM subfamily, with 10 or more LRRs, and the "Short" LRIMs, with 6 or 7 LRRs, also share the signal peptide, cysteine residue patterning, and coiled-coil sequence features of LRIM1 and APL1C. The "TM" LRIMs have a predicted C-terminal transmembrane region, and the "Coil-less" LRIMs exhibit the characteristic LRIM sequence signatures but lack the C-terminal coiled-coil domains.</p> <p>Conclusions</p> <p>The evolutionary plasticity of the LRIM LRR domains may provide templates for diverse recognition properties, while their coiled-coil domains could be involved in the formation of LRIM protein complexes or mediate interactions with other immune proteins. The conserved LRIM cysteine residue patterns are likely to be important for structural fold stability and the formation of protein complexes. These sequence-structure-function relations of mosquito LRIMs will serve to guide the experimental elucidation of their molecular roles in mosquito immunity.</p

Anopheles gambiae blood feeding Initiates an anticipatory defense response to plasmodium berghei

Mosquitoes have potent innate defense mechanisms that protect them from infection by diverse pathogens. Much remains unknown about how different pathogens are sensed and specific responses triggered. Leucine-Rich repeat IMmune proteins (LRIMs) are a mosquito-specific family of putative innate receptors. Although some LRIMs have been implicated in mosquito immune responses, the function of most family members is largely unknown. We screened &lt;i&gt;Anopheles gambiae &lt;/i&gt;LRIMs by RNAi for effects on mosquito infection by rodent malaria and found that LRIM9 is a &lt;i&gt;Plasmodium berghei &lt;/i&gt;antagonist with phenotypes distinct from family members LRIM1 and APL1C, which are key components of the mosquito complement-like pathway. LRIM9 transcript and protein levels are significantly increased after blood feeding but are unaffected by &lt;i&gt;Plasmodium &lt;/i&gt;or midgut microbiota. Interestingly, LRIM9 in the hemolymph is strongly upregulated by direct injection of the ecdysteroid, 20-hydroxyecdysone. Our data suggest that LRIM9 may define a novel anti-&lt;i&gt;Plasmodium &lt;/i&gt;immune defense mechanism triggered by blood feeding and that hormonal changes may alert the mosquito to bolster its defenses in anticipation of exposure to blood-borne pathogens.</jats:p

Large-Scale Modelling of the Environmentally-Driven Population Dynamics of Temperate Aedes albopictus (Skuse)

The Asian tiger mosquito, Aedes albopictus, is a highly invasive vector species. It is a proven vector of dengue and chikungunya viruses, with the potential to host a further 24 arboviruses. It has recently expanded its geographical range, threatening many countries in the Middle East, Mediterranean, Europe and North America. Here, we investigate the theoretical limitations of its range expansion by developing an environmentally-driven mathematical model of its population dynamics. We focus on the temperate strain of Ae. albopictus and compile a comprehensive literature-based database of physiological parameters. As a novel approach, we link its population dynamics to globally-available environmental datasets by performing inference on all parameters. We adopt a Bayesian approach using experimental data as prior knowledge and the surveillance dataset of Emilia-Romagna, Italy, as evidence. The model accounts for temperature, precipitation, human population density and photoperiod as the main environmental drivers, and, in addition, incorporates the mechanism of diapause and a simple breeding site model. The model demonstrates high predictive skill over the reference region and beyond, confirming most of the current reports of vector presence in Europe. One of the main hypotheses derived from the model is the survival of Ae. albopictus populations through harsh winter conditions. The model, constrained by the environmental datasets, requires that either diapausing eggs or adult vectors have increased cold resistance. The model also suggests that temperature and photoperiod control diapause initiation and termination differentially. We demonstrate that it is possible to account for unobserved properties and constraints, such as differences between laboratory and field conditions, to derive reliable inferences on the environmental dependence of Ae. albopictus populations

Transcriptional silencing and activation of paternal DNA during Plasmodium berghei zygotic development and transformation to oocyst

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

Differential effects of azithromycin, doxycycline and co-trimoxazole in ingested blood on the vectorial capacity of malaria mosquitoes

Background.  The gut microbiota of malaria vector mosquitoes grows after a blood meal and limits Plasmodium infection. We previously showed that penicillin and streptomycin in the ingested blood affect bacterial growth and positively impact mosquito survival and permissiveness to Plasmodium. In this study, we examine the effects of doxycycline, azithromycin, and co-trimoxazole. All 3 antibiotics are used in mass drug administration programs and have antimicrobial activities against bacteria and various stages of malaria parasites. Methods.  The effects of blood meal supplementation with antibiotics on the mosquito microbiota, lifespan, and permissiveness to Plasmodium falciparum were assessed. Results.  Ingestion of any of the 3 antibiotics significantly affected the mosquito microbiota. Azithromycin decreased P falciparum infection load and mosquito lifespan, whereas at high concentrations, doxycycline increased P falciparum infection load. Co-trimoxazole negatively impacted infection intensity but had no reproducible effect on mosquito lifespan. Conclusions.  Our data suggest that the overall effect of antibiotic treatment on parameters critical for mosquito vectorial capacity is drug specific. The negative effect of azithromycin on malaria transmission is consistent with current efforts for disease elimination, whereas additional, larger scale investigations are required before conclusions can be drawn about doxycycline

The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito Anopheles gambiae

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1(cut) that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals

Toward the definition of efficacy and safety criteria for advancing gene drive-modified mosquitoes to field testing

Mosquitoes containing gene drive systems are being developed as complementary tools to prevent transmission of malaria and other mosquito-borne diseases. As with any new tool, decision makers and other stakeholders will need to balance risks (safety) and benefits (efficacy) when considering the rationale for testing and deploying gene drive-modified mosquito products. Developers will benefit from standards for judging whether an investigational gene drive product meets acceptability criteria for advancing to field trials. Such standards may be formalized as preferred product characteristics and target product profiles, which describe the desired attributes of the product category and of a particular product, respectively. This report summarizes discussions from two scientific workshops aimed at identifying efficacy and safety characteristics that must be minimally met for an investigational gene drive-modified mosquito product to be deemed viable to move from contained testing to field release and the data that will be needed to support an application for first field release

The peptidoglycan recognition proteins PGRPLA and PGRPLB regulate Anopheles immunity to bacteria and affect infection by Plasmodium

Peptidoglycan recognition proteins (PGRPs) form a family of immune regulators that is conserved from insects to mammals. In the malaria vector mosquito Anopheles coluzzii , the peptidoglycan receptor PGRPLC activates the Imd pathway limiting both t he microbiota load and Plasmodium infection. Here, we carried out an RNAi screen to examine the rol e of all seven Anopheles PGRPs in infections with Plasmodium berghei and Plasmodium falciparum . We show that, in addition to PGRPLC, PGRPLA and PGRPS2/S3 also participate in antiparas itic defenses, and that PGRPLB promotes mosquito permissiveness to P. falciparum . We also demonstrate that following a mosquito blood feeding, which promotes growth of the gut microbiota, PGRPLA and PGRPLB positively and negatively regulate the activation of the Imd pathway, respective ly. Our data demonstrate that PGRPs are important regulators of the mosquito epithelial immunity and vector comp etence