<i>A. gambiae</i> G3 mosquitoes intrathoracically inoculated with 5′ONNVic-eGFP.

<p>Mosquitoes were inoculated with ∼1650 PFU of 5′ONNVic-eGFP. (<b>A</b>) 10 inoculated mosquitoes were collected daily and qrt-PCR was used to ascertain viral genome copy number/mosquito. (<b>B</b>) Percent of inoculated mosquitoes showing GFP expression at 1, 4 and 9 DPI. (<b>C</b>) Brightfield (BF) and fluorescent (GFP) images of GFP expression in the head tissues through the ommatidia (O) of inoculated mosquitoes at 9 DPI and (<b>D</b>) Brightfield (BF) and fluorescent (GFP) images of GFP expression in nerves and/or muscle bands in the anterior- (A) and mid-gut (M) of inoculated mosquitoes. Error bars represent standard deviation of 3 biological replicates.</p

RNAi and qrt-PCR based screening of 19 genes for antiviral function in <i>A. gambiae</i> mosquitoes.

<p>Mosquitoes were injected with dsRNA and ∼3000 PFU of 5′ONNVie-eGFP concurrently, 30 mosquitoes were collected at 7 DPI, and qrt-PCR was used to ascertain viral genome copy number/mosquito. Values were normalised relative to the <i>LacZ</i> (non-specific) control and are given as a percentage relative to the LacZ titre. <i>nsP3</i>, a viral gene was included in the screen. Genes are divided into functional categories based on which immune signalling pathway they belong to, or their putative function. Genes with an asterisk* were selected for further characterisation.</p

Transcriptional responses of <i>A. gambiae</i> G3 mosquitoes to 5′ONNVic-eGFP infection.

<p>The transcriptional responses of <i>A. gambiae</i> mosquitoes inoculated with ≈1640 PFU of 5′ONNVic-eGFP infection were profiled using 4X44K Agilent RNA microarrays. Gene lists include only features that pass strict criteria outlined in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001565#s2" target="_blank">materials and methods</a>. Genes included the analysis are 2-fold or greater regulated at a minimum of 1 of the 3 time points, with T-test P values of <0.05. Genes were categorised based on gene ontology.</p

Four antagonists of 5′ONNVic-eGFP in <i>A. gambiae</i> mosquitoes.

<p>Mosquitoes were injected with dsRNA and ∼3000 PFU/mosquito P1/P2V 5′ONNVic-eGFP concurrently. Individual mosquitoes were collected and subject the plaque assay at 7 DPI. Data was Log10 transformed. Bars represent the median from two independent biological replicates. P values indicate significance from Man Whitney U Testing of each gene KD compared to the LacZ control (P<0.05*, P<0.01**, P<0.001***).</p

Optimization of Armored Spherical Tanks for Storage on the Lunar Surface

<div><p>Reverse genetics in the mosquito <em>Anopheles gambiae</em> 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 <em>An. gambiae</em> 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.</p> </div

<i>In vivo</i> phagocytosis assay.

<p>(A) Pictures of abdominal segments of adult female mosquitos injected with dsLacZ, dsAGAP004928 and dsAGAP008492 and then challenged with pHRodo <i>E. coli</i> bioparticles. Fluorescence and brightfield images were captured and hemocytes containing fluorescent bioparticles quantified. The experiment was repeated twice with 8–10 mosquitoes per treatment. Quantification of fluorescent bioparticles and statistical analysis were performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003145#s3" target="_blank">Materials and Methods</a>. (B) The graph shows the percentage of phagocytosis of the different KDs placing dsLacZ as reference. Mean values and standard errors are reported. Asterisks indicate statistical significance (**: 0,005LacZKD.</p

Bacterial phagocytosis screen.

<p>(A) Venn diagram showing the results of the z-score and ANOVA analyses of data obtained with the microplate reader phagocytosis assay. (B) Heat map depicting the performance in the microscopy imaging and <i>in vivo</i> phagocytosis assays of the 13 dsRNAs identified as positive hits with the microplate reader assay. Dark green, significant decrease of phagocytosis; green, decrease of phagocytosis; dark red, significant increase of phagocytosis; red, increase of phagocytosis; grey, similar to LacZ control; nd, not determined. (C) Microscopy imaging analysis: phagocytosis rates of cells at 2 and 20 h following bioparticles challenge when genes identified as modulators of phagocytosis by microplate reader assay are silenced. Data are shown as percent phagocytosis compared to ds<i>LacZ</i>-treated controls. (D) Examples of fluorescence microscopy images of Sua5.1* cells treated with dsRNAs or Cytochalasin D. Images were captured 20 h after challenge with pHRodo bioparticles. The graphs indicate the capacity of cells to uptake bioparticles following ds<i>LacZ</i>, ds<i>Cactus</i> and Cytochalasin D treatments at 2 and 20 h after challenge, as quantified by image analysis. Mean values of counted particles and standard errors are reported. Results from two experiments are shown. Asterisks indicate statistically significant differences between each KD and the dsLacZ-treated controls (*: P<0,05; **: 0,005</p

Luciferase assays.

<p>(A) Transcriptional activation of <i>CEC1</i> promoter upon PGN challenge. The graph reports the z-scores calculated from the ratios of averaged RLU measurements after PGN and PBS treatments. (B and C) Transcriptional regulation of LRIM1 promoter (B) upon PGN challenge (z-scores calculated from the ratios of averaged RLU measurements after PGN and PBS treatments) and (C) in basal conditions (z-scores calculated from the RLU measured after PBS treatments). Positive hits are shown as open circles.</p

Schematic representation of the role of identified regulators in the <i>An. gambiae</i> hemocyte immune response.

<p>A total of 22 positive (green) and negative (red) regulators are shown. Dotted lines indicate modulations of <i>CEC1</i> and <i>LRIM1</i> gene expression upon PGN challenge, and solid lines describe the effect of genes on phagocytosis and basal expression of <i>LRIM1</i>.</p

Cell growth and viability screen.

<p>(A) A fraction of the dsRNA hemocyte-specific library was screened twice by automated fluorescence microscopy. Fluorescent cells were automatically counted using protocols developed in Volocity Improvision and ImageJ software, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003145#s3" target="_blank">Materials and Methods</a>. The ratios of dead cells (Sytox Green positive) over the total number of cells (Hoechst positive), and the standard deviation of replicates, are shown. (B) The entire dsRNA collection was screened using the Cell Titer Blue/Plate Reader method. The plot shows the z-score analysis of one representative set of experiments. A z-score threshold of at least 2 was chosen, and positive hits are shown as open circles. No dsRNA-treated samples and samples treated with 100 mM H₂O₂ are also indicated. Three biological replicates were performed.</p