Effect of AgMC1 silencing on <i>A. gambiae</i> susceptibility to <i>P. berghei</i> infection.

<p>(A) AgMC1 midgut mRNA levels in sugar-fed <i>A. gambaie</i> G3 females 2 days after injection of dsLacZ (control) or dsAgMC1. Effect of AgMC1 silencing on the number of <i>Plasmodium berghei</i> oocysts in midguts dissected (A) 8 days or (B) 2 days post feeding. Effect of AgMC1-silencing on the number of live (green dots, left panel) or melanized (black dots, right panel) (C) and prevalence (D) of <i>P. berghei</i> infection in <i>A. gambiae</i> refractory (L35 strain) females 7 days post-infection. Medians are indicated by the red lines and distributions were compared using the Kolmogorov-Smirnov test. (***indicates <i>P</i><0.001; Student’s t test).</p

Effect of AgMC1 silencing on mitochondrial ROS generation in the midgut.

<p>(A) Control (dsLacZ) and dsAgMC1-silenced midguts from sugar-fed mosquitoes were incubated with dihydroethidine (DHE), a dye that becomes fluorescent in response to ROS. DIC images of the same midguts are shown in the inset. Scale bar  = 100 µm. (B) Effect of AgMC1 silencing, co-silencing Duox (C) or NOX5 (D) or adding FCCP (E) on midgut H₂O₂ generation detected using the Amplex red assay. Resorufin fluorescence was measured at 590 nm. Bars represent means ± SE. Significant differences are indicated by the asterisks (***indicates <i>P</i><0.001; Student’s <i>t</i>-test).</p

<i>Anopheles gambiae</i> mitochondrial carrier 1 (AgMC1) phylogeny and predicted structure.

<p>(A) Phylogenetic tree based on the sequence alignment of the deduced amino acid sequence of AgMC1 (AGAP001297-PA) and the putative mitochondrial carriers from <i>A. aegypti</i> (AaMC1), <i>D. melanogaster</i> (DmMC1), human mitochondrial carriers HsSLC25A-39 and SLC25A-40 and yeast manganese trafficking factor for mitochondrial (ScMTM1); uncoupling proteins from humans (HsUCP), <i>A. gambiae</i> (AgUCP), <i>A. aegypti</i> (AaUCP) and <i>D. melanogaster</i> (DmUCP); putative adenine nucleotide translocators (ANT) from humans (HsANT, SLC25A6), yeast (ScANT), <i>A. gambiae</i> (AgANT) <i>A. aegypti</i> (AaANT), and <i>D. melanogaster</i> (DmANT); putative phosphate carriers (PiC) from humans (HsPiC, SLC25A3), yeast (ScPiC), <i>A. gambiae</i> (AgPiC), <i>A. aegypti</i> (AaPiC,) and <i>D. melanogaster</i> (DmPiC). Sequence alignments and accession numbers are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041083#pone.0041083.s001" target="_blank">Fig. S1</a>. (B) Schematic representation of the AgMC1 protein sequence coding for three mitochondrial carrier domains (mito carr, top panel) highlighted in blue, green and red, and of the predicted secondary structure (bottom panel), consisting of six transmembrane domains (H1 to H6), three matrix domains, (M1 to M3), and cytosolic domains. (E) Predicted tertiary structure based on the amino acid sequence of the AgMC1 based on the known structure of bovine ADP/ATP adenine nucleotide translocator. Ribbon diagram of the predicted structure of AgMC1 from a lateral view (left), or viewed from either the matrix (top right) or intermembrane space side of the mitochondrial membrane (bottom right).</p

Effect of AgMC1 silencing on midgut mitochondrial respiration and coupling.

<p>(A) Oxygen consumption of midguts from sugar-fed <i>A. gambiae</i> females injected with dsLacZ or dsAgMC1 after the addition of ADP, oligomycin (oligo), or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (B) RCR from paired groups of <i>A. gambiae</i> midguts. The <i>A. gambiae</i> respiration data represent seven biological replicates from three independent experiments. (C) Oxygen consumption of midguts from sugar-fed <i>A. aegypti</i> females injected with dsLacZ or dsAaMC1 after the addition of ADP, oligomycin (oligo), or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (D) RCR from paired groups of <i>A. aegypti</i> midguts. The <i>A. aegypti</i> respiration data represent four biological replicates from two independent experiments. (E) Effect of AgMC1 silencing midgut on mitochondrial membrane potential. Confocal analysis of control (dsLacZ) and silenced (dsAgMC1) midguts stained with MitoTracker Red (CM-H₂-XRos) to evaluate membrane potential. Nuclei were stained with DAPI (blue) and F-actin with green phalloidin. Scale bar  = 8 µm. (*and **indicate <i>P</i><0.05 and P<0.001, respectively; ANOVA). The RCR values were compared using the paired T–test.</p

Catalase mRNA and activity increased in the midgut epithelia of blood-fed mosquitoes.

<p><i>Aedes aegypti</i> females were fed sugar (SF) or blood (BF) and dissected at the indicated time points after a blood meal. (A) Catalase mRNA expression in the epithelia was evaluated using qPCR analysis of SF and BF mosquitoes. (B) Enzymatic activity was measured as described in the Materials and Methods section. (C) Catalase inhibition by AT <i>in vitro</i>. Midgut epithelia was collected from blood-fed mosquitoes 24 h after feeding and was incubated with AT and H₂O₂ for 30 min at 4°C; then, catalase activity was assayed. (D) Mosquitoes were fed blood supplemented with 15 mM AT and assayed for catalase activity in the epithelia *** <i>p <</i> 0.001. Figure 1A-B–ANOVA followed by Dunnett's multiple comparison test.</p

Catalase knockdown in the midgut.

<p>(A) Two-day-old females were injected with dsRNA against catalase (dsCat) or an unrelated control gene (dsLac). Two days after the dsRNA injection, a group of mosquitoes were fed blood, while others were fed exclusively with sugar. Twenty-four hours later, RNA was extracted for qPCR analysis. (B) Catalase activity in the epithelia was measured 24 h after a blood meal in dsRNA-treated mosquitoes. (C) Hydrogen peroxide leakage was measured in the midgut epithelia of SF mosquitoes injected with dsCat or dsLacZ. (A) *** <i>p <</i> 0.001 (t-test). (B) **** <i>p <</i> 0.0001 (t-test). (C) * <i>p</i> = 0.0404 (t-test).</p

Catalase knockdown in the midgut.

<p>(A) Two-day-old females were injected with dsRNA against catalase (dsCat) or an unrelated control gene (dsLac). Two days after the dsRNA injection, a group of mosquitoes were fed blood, while others were fed exclusively with sugar. Twenty-four hours later, RNA was extracted for qPCR analysis. (B) Catalase activity in the epithelia was measured 24 h after a blood meal in dsRNA-treated mosquitoes. (C) Hydrogen peroxide leakage was measured in the midgut epithelia of SF mosquitoes injected with dsCat or dsLacZ. (A) *** <i>p <</i> 0.001 (t-test). (B) **** <i>p <</i> 0.0001 (t-test). (C) * <i>p</i> = 0.0404 (t-test).</p

Catalase silencing impacted Dengue but not Zika midgut infection prevalence.

<p>(A) Females were fed blood contaminated with 10⁷ PFU/mL of Zika virus, and 7 days after feeding the number of PFU was determined in the midgut. (B) The percentage of infected midguts (infection prevalence) was scored from the same set of data as in A. (C) Mosquitoes were fed a lower dose of Zika-infected blood (10⁵/mL), and PFU/midgut was determined 7 days post-infection. (D) Infection prevalence of mosquitoes from C. (E) Mosquitoes were fed blood contaminated with 10⁶ PFU/mL of Dengue 4 virus, and PFU/midgut was counted 7 DPI. (F) Infection prevalence was determined from the same group of mosquitoes. Mann-Whitney <i>U</i>-tests were used for infection intensity (A, C, E), and chi-square tests were performed to determine the significance of infection prevalence analysis (B, D, F). Statistical values and number of replicates are depicted in the corresponding figures.</p

Catalase knockdown affected both resistance to H₂O₂ and oviposition.

<p>Catalase was silenced as described, and two days after dsRNA injection, SF (A) or BF (immediately after feeding) (B) mosquitoes were transferred to cages containing 5% sucrose supplemented with 1 M H₂O₂ <i>ad libitum</i> (day 0). A fresh H₂O₂ solution was provided daily. Survival was scored every 24 h. *** <i>p</i> < 0.0001 for the comparison between dsLacZ–H₂O₂ vs dsCat–H₂O₂ (log-rank test). (C) Catalase-silenced mosquitoes were blood-fed and allowed to lay eggs. Each dot represents an individual mosquito. LacZ–n = 21. Catalase–n = 25. * <i>p =</i> 0.37 (t-test).</p