Lectin-blot and immuno-blot analysis of <i>L. longipalpis</i> salivary glycoproteins.

<p>Effect of enzymatic deglycosylation with PNGase F on two sources of sand fly salivary glycoproteins, SGL and HSL. Samples were stained for protein composition profile (Imperial), probed for the presence of N-linked glycans (GNL, AAL) and O-linked glycans (VVL, PNA), or probed for reactivity with IgG antibodies of animals exposed to the bite of sand flies on BluePort-associated skin. Arrows indicate the main antigenic salivary glycoproteins.</p

Inflammatory response of naïve and immune mice to sand fly bites on normal skin.

<p>Inflammatory response detected 48 hours after exposure to the bite of <i>L. longipalpis</i> sand flies on naïve mice (A and B) and immune mice (C and D). Black arrows indicate neutrophils and white arrows eosinophils. Hematoxylin-eosin stain. Magnification, 200x in (A and C), 1000X in (B and D).</p

Chronic inflammatory response to CBa-beads.

<p>Histopathological changes observed 30 days (A and B), 60 days (C and D), 90 days (E and F) and 120 days (G and H) days after injection of CBa-beads. Arrows indicate endothelial cells in (A), macrophages in (B), neutrophils inside a blood vessel in (C), capsule in (D), multinucleated foreign-body giant cells in (E), mast cells in (F), eosinophils in (G) and lymphocytes in (H). Hematoxylin-eosin stain, 1000x magnification.</p

Localization and appearance of the BluePort tissue compartment.

<p>Macroscopic appearance of the blue nodule (BluePort) formed one month after injection of CBa-beads in the subcutaneous tissue of mice (A and B). Macroscopic (C) and microscopic appearance (D) of BluePort-associated skin 24 hours after exposure of naïve mice to the bite of <i>L. longipalpis</i> sand flies. Black arrows indicate skin erythema in (C), and vasodilatation of dermal blood vessels in (D). White arrow indicates vasodilatation of blood vessels inside the BluePort parenchyma in (D). Hematoxylin-eosin stain, magnification 100x in (D).</p

Inflammatory response in mice exposed twice to sand fly bites on BluePort-associated skin.

<p>Evolution of the inflammatory response in samples collected 24 hours (A, E and I), 48 hours (B, F and J), 72 hours (C, G and K) and 96 hours (D, H and L) after exposure for the second time to the bite of sand flies. Black arrows indicate neutrophils and white arrows indicate eosinophils. Hematoxylin-eosin stain. Magnification: 100x in (A–D), 1000x in dermis (E–H) and hypodermis (I–L).</p

<i>w</i> ratios for all nucleotides mapped across annotated Dicer genes.

<p>The amino-terminal domains of most Dicer enzymes contain a DExH-box RNA or ERCC4-like helicase domain followed by members of helicase superfamily c-terminal domains containing a number of nucleotide binding regions (gray ovals) and an ATP binding domain on Dcr 2 (black ovals). One or two double stranded RNA-binding domains (dsRBDs) consisting of ∼100 amino acid residues occur in the center and the carboxyl end of Dicers. The first of these dsRBDs is followed by the oligonucleotide-binding (indicated with vertical lines) PAZ domain located in the center of Dicer where it binds the 5′ phosphates and 2 nt 3′ hydroxyl overhangs. Cleavage is accomplished by dimerized RNase III domains (labeled RIBOc). Active sites (black ovals) are shown on Dcr2. The areas of dimerization are labeled with ‘*’ while regions of metal ion-binding are labeled with a ‘+.’</p

<i>Aedes aegypti</i> collection sites.

<p><i>Aedes aegypti</i> collection sites.</p

Intraspecific patterns in miRNA and siRNA pathway genes of <i>Ae. aegypti</i>.

<p>Sample size (N), numbers of segregating sites that were synonymous (S_s) or led to amino acid replacements (S_a), the number of haplotypes (h), the overall pi(the average number of nucleotide differences per site between two sequences) are listed for each gene and collection. Also listed are the average number of nucleotide differences, k and K among synonymous sites (K_s) and among replacement sites (K_a) and their ratio (K_a/K_s). The effective recombination rate between adjacent sites (R) and the PHI test for recombination are listed alongside Fu and Li's F* test and associated significance tests (*P<0.05, **P<0.01).</p

Phenotype correlation analysis.

<p>To identify potential correlations of Ae aegypti susceptibility to dengue virus infection with measures of nucleotide and amino acid diversity in RNAi genes, Pearson correlation analyses were performed between DENV-2 MIB (the proportion of orally virus-exposed female mosquitoes that fail to develop a midgut infection) and MEB (the proportion of females with a midgut infection that fail to develop a disseminated infection) with p (number of nucleotide differences per site between all sequence pairs), k (number of nucleotide differences between all sequence pairs), K_A (number of nucleotide differences among replacement sites), K_S (number of nucleotide differences among synonymous sites) and K_A/K_S. Pearson correlation coefficients are shown.</p>*<p>P≤0.05,</p>**<p>P≤0.01.</p

Primers for PCR amplification of most of the exon regions of <i>dcr1</i>, <i>dcr2</i>, <i>ago1</i>, <i>ago2</i>, <i>r2d2</i> and <i>r3d1</i>.

<p>Primer locations are numbered with respect to supercontigs in the <i>Ae. aegypti</i> genome project in Vector Base (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044198#pone-0044198-g002" target="_blank">Figure 2</a>). An * indicates that all or part of that primer is located in an intron.</p