Resistance of genetically modified mosquitoes to insecticides.

<p>Bloodfed adult female mosquitoes were exposed to various insecticides for 1 h immediately following a blood meal, and their survival was recorded A) 1 h or B) 24 h post-exposure. Genetically modified mosquitoes showed no difference from wild-type mosquitoes in their resistance to various insecticides. Each figure represents 25 mosquitoes from each of 3 generations.</p

Genetic modification of <i>Anopheles stephensi</i> for resistance to multiple <i>Plasmodium falciparum</i> strains does not influence susceptibility to o’nyong’nyong virus or insecticides, or Wolbachia-mediated resistance to the malaria parasite

<div><p>Mosquitoes that have been genetically engineered for resistance to human pathogens are a potential new tool for controlling vector-borne disease. However, genetic modification may have unintended off-target effects that could affect the mosquitoes’ utility for disease control. We measured the resistance of five genetically modified <i>Plasmodium</i>-suppressing <i>Anopheles stephensi</i> lines to o’nyong’nyong virus, four classes of insecticides, and diverse <i>Plasmodium falciparum</i> field isolates and characterized the interactions between our genetic modifications and infection with the bacterium <i>Wolbachia</i>. The genetic modifications did not alter the mosquitoes’ resistance to either o’nyong’nyong virus or insecticides, and the mosquitoes were equally resistant to all tested <i>P</i>. <i>falciparum</i> strains, regardless of <i>Wolbachia</i> infection status. These results indicate that mosquitoes can be genetically modified for resistance to malaria parasite infection and remain compatible with other vector-control measures without becoming better vectors for other pathogens.</p></div

Resistance of genetically modified mosquitoes to ONNV.

<p>Adult female mosquitoes were provided an infectious blood meal containing O’nyong’nyong virus (ONNV). Genetically modified mosquitoes showed no difference from wild-type mosquitoes in their ability to be infected with ONNV. This figure represents 10 mosquitoes from each of 3 generations compared by a Kruskal-Wallis test, with α = 0.05. Additional data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195720#pone.0195720.s002" target="_blank">S2 Table</a>.</p

Resistance of genetically modified mosquitoes to multiple <i>P</i>. <i>falciparum</i> strains.

<p>Adult female mosquitoes were fed on infectious blood meals containing gametocytes from the A) Kenyan HL1204 and B) Brazilian 7g8 <i>P</i>. <i>falciparum</i> strains. All genetically modified mosquito strains tested exhibited increased resistance to both <i>P</i>. <i>falciparum</i> strains, indicating that these mosquitoes resist <i>P</i>. <i>falciparum</i> from multiple, geographically divergent, areas. * indicates a significant difference in the number of oocysts per midgut when compared to wild-type mosquitoes using a Kruskal-Wallis test, followed by a Dunn’s post-hoc test and α = 0.05. Additional data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195720#pone.0195720.s001" target="_blank">S1 Table</a>.</p

Cytoplasmic Actin Is an Extracellular Insect Immune Factor which Is Secreted upon Immune Challenge and Mediates Phagocytosis and Direct Killing of Bacteria, and Is a <i>Plasmodium</i> Antagonist

<div><p>Actin is a highly versatile, abundant, and conserved protein, with functions in a variety of intracellular processes. Here, we describe a novel role for insect cytoplasmic actin as an extracellular pathogen recognition factor that mediates antibacterial defense. Insect actins are secreted from cells upon immune challenge through an exosome-independent pathway. <i>Anopheles gambiae</i> actin interacts with the extracellular MD2-like immune factor AgMDL1, and binds to the surfaces of bacteria, mediating their phagocytosis and direct killing. Globular and filamentous actins display distinct functions as extracellular immune factors, and mosquito actin is a <i>Plasmodium</i> infection antagonist.</p></div

Actin is an antagonist of <i>Plasmodium</i> infection.

<p><i>P. falciparum</i> oocyst–stage infection intensity after silencing of (A) <i>An. gambiae actin (Ac)</i>, <i>AgMDL1</i> (MDL1), <i>actin</i> and <i>AgMDL1</i> (Ac+MDL1), or GFP or (B) <i>actin</i> (Ac) or <i>GFP</i> in septic vs aseptic mosquitoes. Circles represents the number of oocysts in an individual mosquito midgut, and the horizontal line indicates the median number. Three independent replicates were obtained, and the Mann-Whitney test was used to determine statistical significance and p-values (indicated above each group).</p

Actin is secreted into the cell culture supernatant fraction upon immune challenge via an exosome independent mechanism that is regulated by immune pathways.

<p>(A) Immune-challenged (LPS-Pa, LPS-EcK12, LPS-Ec; 10 μg/mL Lys-PGN; 20 μg/mL, or DAP-PGN; 1 μg/mL) Sua5B (<i>An. gambiae</i>), and S2 (<i>D. melongaster</i>) insect cell supernatants and soluble lysate fractions examined for the presence of actin (upper panel) or tubulin (lower panel). (B) Viability of Sua5B and S2 cell lines after treatment with LPS-Pa (10 μg/mL), Lys-PGN (20 μg/mL) or DAP-PGN (1 μg/mL) for 24 hr was determined using the Cell Titer Fluor Cell Viability Assay. (C) Cell viability of Sua5B and S2 cells after saponin (1–10⁻⁶%)-induced cell lysis and (D) the amount of actin released into the supernatant was determined for Sua5B (upper panel) and S2 cells (lower panel) at varying saponin concentrations (10⁻⁵, 10⁻⁴, 10⁻³ and 1%). (E) Supernatant fractions of Sua5B and Moss55 cells challenged with LPS (10 μg/mL) or Lys-PGN (20 μg/mL) for 24 hr in the presence of 5 μM GW4869 or DMSO (control) and probed for actin. (F) Exosomes (EXO) and lysate fractions isolated from LPS and Lys-PGN stimulated Sua5B or Moss55 cells analyzed for actin. (G) Supernatant fraction of <i>caspar</i> and <i>cactus</i> silenced Sua5B cells probed for the presence of actin.</p

Globular and filamentous actin display different immune properties.

<p>(A) Actin content in 0.3–0.5M NaCl eluates (E3–E5 fractions) or pellets (P) of <i>E. coli</i> and <i>S. aureus</i> incubated with recombinant globular (G Ac) or filamentous (F Ac) actin. (B) FITC-labeled <i>E. coli</i> (green) incubated with LPS-stimulated Sua5B cell supernatants and stained for AgMDL1 (blue) and actin (red). Co-localization is indicated in white. (C) The percentage of phagocytosing S2 cells containing at least one <i>E. coli</i> bacterium incubated with recombinant globular (G Ac) or filamentous (F Ac) actin with or without AgMDL1 or the control protein GUS as compared to untreated bacteria (PBS). For each assay, at least 16 fields were counted, and the data are representative of three independent experiments. Each bar represents the mean ± the standard deviation. Statistical significance was determined using Student’s <i>t</i>-test. (D) Viability of <i>E. coli</i> was determined after 1- or 24-hr incubations with recombinant globular (G Ac) or filamentous actin (F Ac), with or without AgMDL1, or the control protein GUS as compared to untreated bacteria (PBS). Error bars represent the mean ± the standard deviation. Statistical significance was determined using Student’s <i>t</i>-test. (E) Scanning electron microscopy image of <i>E. coli</i> cells after incubation with G actin (G Ac), F actin (F Ac), AgMDL1 (MDL1) alone or together (G Ac+MDL1, F Ac+MDL1) or with the control protein GUS. Untreated cells were incubated with PBS. Scale bar 200 nm.</p

<i>An. gambiae</i> actins display tissue, developmental-specific, and immune-responsive expression.

<p>Transcript abundance of all <i>An. gambiae actin</i> genes in the (A) thorax, abdomen, and midgut adult tissues or (B) at distinct developmental stages compared to the adult female mosquito and normalized using the <i>An. gambiae</i> ribosomal <i>S7</i> gene. Actin 5C (651 A, B, C); larval muscle actin (5095); adult muscle actin (1516); minor actins (1676) and (2127). Expression of actin 5C (651 A, B, C) and adult muscle actin (1516) in <i>An. gambiae</i> Sua5B cells after challenge with (C) LPS (10 μg/mL) (D) Lys-PGN (20 μg/mL) or (E) DAP-PGN (1 μg/mL) for 1,3,6 and 24 hr or (F) live <i>E. coli</i> and <i>S. aureus</i> (MOI 100) for 30 min or 2 hr, compared to non-challenged cells and normalized using the <i>An. gambiae S7</i> gene.</p

Actin is an extracellular immune factor.

<p>(A) Incubation of <i>E. coli</i> and <i>S. aureus</i> with actin (Ac), AgMDL1 (MDL1), actin +AgMDL1 (Ac+MDL1) or the control protein GUS. Untreated bacteria were incubated with PBS. The percentage of phagocytosing cells was calculated as the number of S2 cells containing at least one phagocytized FITC-labeled bacterium compared to the total number of cells in the field. For each experiment at least 16 fields were counted and the data are representative of three independent experiments. Each bar represents the mean ± the standard deviation. Statistical significance was determined using Student’s <i>t</i>-test.(B) Fluorescent microscopy of S2 cells (red) and FITC-labeled <i>E. coli</i> or <i>S. aureus</i> bacteria (green) incubated with the recombinant proteins actin (Ac) or AgMDL1 (MDL1) alone, or both together (Ac+MDL1) or the control protein GUS. Untreated bacteria were incubated with PBS. (C) Viability of <i>E. coli</i> and <i>S. aureus</i> was determined after 1 or 24 hr of incubation with recombinant proteins (Ac, MDL1, Ac+MDL1, GUS) and compared to untreated bacteria. Error bars represent the mean ± the standard deviation. Statistical significance was determined using Student’s <i>t</i>-test. (D) <i>An. gambiae</i> survival rates after silencing of <i>actin</i>, <i>AgMDL1</i>, <i>actin</i> and <i>AgMDL1</i>, or <i>GFP</i> (control) and challenge of female mosquitoes with <i>E. coli</i> (OD 1.5) or <i>S. aureus</i> (OD 0.4) four days later. Three biological experiments were performed and combined and statistical analysis consisted of a log-rank test to determine the overall significance between all groups, followed by pairwise comparisons between <i>GFP</i> and the other three groups. (E) <i>An. gambiae</i> thorax (6 μg), abdomen (6 μg) and midgut (12 μg) tissues along with hemolymph (2 μg) fractions challenged with LPS (100ng) for 4 hr analyzed for the presence of actin 4 days after silencing of <i>actin</i> or <i>GFP</i> (control). (F) Hemolymph extract from adult female <i>An. gambiae</i> probed for the presence of actin after silencing of <i>GFP</i> (control), <i>actin</i> (Ac) or <i>AgMDL1</i> (MDL1).</p