Shin_SupMat_Table 1

Supplementary Table 1. Sex ratios among BC2 progeny from crosses of RED strain females mated with BC1 males from crosses between T37 strain males and RED strain females

Mapping data: two point linkage estimates and standard errors.

Composite two point linkage estimates and standard errors for each linkage group in the Culex pipiens genetic map

Development of the <i>Ae. aegypti</i> embryonic ventral nerve cord.

<p>Anti-acetylated tubulin staining (A–C) marks the developing axon tracts in 52 hr. (A) and 56 hr. (B) <i>Ae. aegypti</i> embryos. By 56 hrs. (C), the <i>Ae. aegypti</i> nerve cord resembles that of a 33 hr. <i>An. gambiae</i> embryo and a St. 16 <i>Dr. melanogaster</i> nerve cord (BP102 staining is shown in D). These time points in the three respective species correspond to germ-band retracted embryos in which segmentation is obvious and organogenesis has initiated. Filleted nerve cords are oriented anterior up in all panels. The anterior commissure is marked by a black arrowhead, and a white arrowhead marks the posterior commissure.</p

Defect in antigen-induced NHR in antigen-specific IgE-Tg mice.

<p>IgE-Tg mice as well as antigen-immunized mice and T cell-transferred mice were challenged 3–4 times with OVA or saline, as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146686#pone.0146686.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146686#pone.0146686.g006" target="_blank">6</a>. Six hours after the 3rd challenge, antigen-specific serum IgE, IgG, IgG₁, IgG_2a, IgG_2b, and IgG₃ were determined (A). The number of sneezes evoked by histamine (B) and accumulation of lymphocytes, neutrophils, and eosinophils in NALF (C) of IgE-Tg mice were examined 6 h after the 4th challenge. Data are expressed as mean ± SEM for 4–8 animals. *<i>p</i> < 0.05, **<i>p</i> < 0.01 (Dunn’s test). N.D.: not detectable.</p

<i>Ae. aegypti fra</i> knockdown CNS phenotypes.

<p>Anti-acetylated tubulin staining (reddish brown) marks the axons of the ventral nerve cords of scrambled control (A) and <i>fra</i> siRNA injected embryos (B–D). Knockdown phenotypes characterized by thinning or loss of commissural axons were observed at 54 hrs. (B–D). Comparable results were obtained with two different siRNAs (<i>fra</i> siRNA-A in B,C; <i>fra</i> siRNA-B in D). Knockdown of <i>fra</i> was confirmed by double-labeling to detect <i>fra</i> mRNA expression (dark blue in C). Nerve cords are oriented anterior up in each panel. The anterior commissure is marked by a black arrowhead, and a white arrowhead marks the posterior commissure.</p

Expression of <i>fra</i> in the developing mosquito CNS.

<p>Comparable <i>fra</i> expression patterns are detected in lateral views of the developing nervous systems (arrows) of <i>An. gambiae</i> (33 hrs., A) and <i>Ae. aegypti</i> (52 hrs., B). Ventral views of <i>Aae fra</i> expression in 52 hr. (C, segments T3–A5) and 54 hr. (D; segments A2–A6) <i>Ae. aegypti</i> embryos are shown. Anterior is oriented left in A and B and up in C and D.</p

Confirmation of <i>fra</i> knockdown in <i>Ae. aegypti.</i>

<p>qRT-PCR was used to assess <i>fra</i> levels following microinjection of <i>fra</i> siRNA-A. A scrambled version of <i>fra</i> siRNA-A was injected as a control. At 72 hrs. post injection, levels of <i>fra</i> were 80% less than that of the control-injected group (N = 3, p<0.0001).</p

Antigen-induced NHR in mast cell-deficient mice.

<p>Immunized W/W^v and +/+ mice were challenged 7 times with OVA or saline, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146686#pone.0146686.g001" target="_blank">Fig 1</a>. Six hours after the last challenge (day 43), the number of sneezes evoked by histamine (A), the accumulation of lymphocytes, neutrophils, and eosinophils in NALF (B), and the antigen-specific serum IgE levels were examined (C). Data are expressed as mean ± SEM for 4–9 animals. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, compared with saline-challenged control mice (Mann-Whitney <i>U</i> test).</p

Antigen-induced NHR in T cell-transferred mice.

<p>Twenty-four hours after transfer of Th1, Th2, or Th17-polarized cells, mice were challenged 3 times with OVA or saline. Six hours after the last challenge, the number of sneezes evoked by histamine (A), the accumulation of lymphocytes, neutrophils, and eosinophils in NALF (B), and the IFN-γ, IL-4, and IL-17 mRNA expression in the nasal tissue (C) were examined. Data are expressed as mean ± SEM for 4–8 animals. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, compared with naïve T cell-transferred and OVA-challenged mice (Dunn’s test). N.D.: not detectable.</p

Quantification of <i>Aae fra</i> knockdown phenotype penetrance and severity.

<p>Embryos stained with anti-acetylated tubulin were scored at 54 hrs. post-injection of <i>fra</i> siRNA-A or scrambled control siRNA. The number and percentage of total segments bearing wild-type (WT), mild, or strong phenotypes in the anterior and posterior commissures are reported. Mild phenotypes correspond to thinning commissures, and severe phenotypes correspond to near or complete absence of commissural axons.</p