IAFGP confers resistance to cold stress in <i>D. melanogaster</i> by preventing apoptotic progress.

<p>(A) Immunoblot analysis using antibodies to caspase -2, -3 and -9 is shown. Total lysates were prepared from <i>iafgp</i>- or <i>mcherry</i>-expressing flies after cold shock at 4°C for 6 days. Untreated flies incubated at 25°C served as experimental controls. Levels of actin served as loading control. (B) ELISA analysis using antibodies to caspase-2, -3 and -9 is shown. Total lysates were prepared from <i>iafgp</i>- or <i>mcherry</i>-expressing flies after cold shock at 4°C for 6 days and coated onto ELISA plates and assayed in triplicates as described in methods. Bovine serum albumin (BSA) was used as control sample. Error bars represent (+) standard deviation from the mean value.</p

Expression of <i>iafgp</i> increases cold tolerance in <i>D. melanogaster</i> adult flies.

<p>Survival of male (A) and female (B) <i>iafgp-or mcherry</i>-expressing flies after cold treatment at 4°C for 2, 4, 6, 7, 8, 9 and 10 days. Mean value from six independent experiments with 25 flies/group/time point is shown. Error bars indicate (±) standard deviation from the mean value. Statistical significance (P<0.05) is indicated with an asterisk and was calculated using ANOVA and Tukey's post test.</p

Generation of <i>iafgp</i>-transgenic <i>D. melanogaster</i>.

<p>(A) Schematic representation of constructs used to generate <i>iafgp</i>-or <i>mcherry</i>-transgenic <i>D. melanogaster</i> is shown. (B) RT-PCR results showing expression of <i>iafgp</i> or <i>mcherry</i> transcripts in <i>iafgp</i>-or <i>mcherry</i>-expressing male (M) and female (F) flies. Reactions performed with no reverse transcriptase enzyme are shown as −RT. Levels of 28S gene transcripts were used as controls. Quantitative PCR results showing levels of <i>iafgp</i> (C) or <i>mcherry</i> (D) transcripts normalized to the levels of 28S gene transcripts in <i>iafgp</i>-or <i>mcherry</i>-expressing male and female flies.</p

Expression of <i>iafgp</i> increases cold tolerance in <i>D. melanogaster</i> embryos.

<p>Quantitative RT-PCR results showing levels of <i>iafgp</i> (A) or <i>mcherry</i> (B) transcripts normalized to actin transcript levels in embryos collected from <i>iafgp-or mcherry</i>-expressing flies. Shown are the results from three independent experiments. (C) Embryos were collected from <i>iafgp-or mcherry</i>-expressing flies after cold treatment at −5°C for 60, 90, 120 and 150 min. Percentage hatching of embryos to larvae was calculated as described in methods. Mean value from eight independent experiments with 20 embryos/group/time point is shown. Error bars indicate (+) standard deviation from the mean value. As controls, embryos incubated at 25°C (untreated) or 4°C for 4 h or cold treated at −5°C for 120 min without 4°C pause were included. Statistical significance (P<0.05) was calculated using ANOVA and Tukey's posttest.</p

Altered vector competence in an experimental mosquito-mouse transmission model of Zika infection

<div><p>Few animal models of Zika virus (ZIKV) infection have incorporated arthropod-borne transmission. Here, we establish an <i>Aedes aegypti</i> mosquito model of ZIKV infection of mice, and demonstrate altered vector competency among three strains, (Orlando, ORL, Ho Chi Minh, HCM, and Patilas, PAT). All strains acquired ZIKV in their midguts after a blood meal from infected mice, but ZIKV transmission only occurred in mice fed upon by HCM, and to a lesser extent PAT, but not ORL, mosquitoes. This defect in transmission from ORL or PAT mosquitoes was overcome by intrathoracic injection of ZIKV into mosquito. Genetic analysis revealed significant diversity among these strains, suggesting a genetic basis for differences in ability for mosquito strains to transmit ZIKV. The intrathoracic injection mosquito-mouse transmission model is critical to understanding the influence of mosquitoes on ZIKV transmission, infectivity and pathogenesis in the vertebrate host, and represents a natural transmission route for testing vaccines and therapeutics.</p></div

Genes proximal to microsatellite clusters driving genetic variability in mosquito strains.

<p>Genes proximal to microsatellite clusters driving genetic variability in mosquito strains.</p

Ho Chi Minh (HCM), Patilas (PAT) and Orlando (ORL) strains of <i>Aedes aegypti</i> are genetically diverse.

<p>Six strains of <i>Ae</i>. <i>aegypti</i> (Liverpool, Rockefeller, Ho Chi Minh (HCM), Orlando (ORL), Amacuzac (Mexico) and Patillas (Puerto Rico) strains) were genotyped at 12 microsatellite loci and population genetic analyses were performed to compare these populations. (A) PCA analysis showing the extent of genetic diversity of various laboratory and field-collected strains of <i>Ae</i>. <i>aegypti</i>. The bar plot with eigenvalues shows the amount of variance represented by each principal component, black bars indicate the components illustrated in these PCA. The units of the grid are indicated at the top right corner. (B) Results of the Bayesian clustering analysis with STRUCTURE [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006350#pntd.0006350.ref024" target="_blank">24</a>]. Shown is the bar plot indicating the genetic groupings of the three strains used for ZIKV infections. Each vertical bar represents an individual. The height of each bar represents the probability of assignment to each of (upper panel) K = 2 clusters or (lower panel) K = 3 clusters. Each cluster is indicated by different colours: HCM: blue, PAT: pink and ORL: red. (C) Frequency of alleles highlighting the differences among <i>Aedes aegypti</i> laboratory populations from Ho Chi Minh, Orlando Strain, and Patilas, challenged with ZIKV. Two of the 12 microsatellite loci genotyped contributed to the population differentiation observed at a loading threshold of 0.10; AC1 and CT2. More specifically, alleles AC1:209, CT2:184, and CT2:188. Alleles are represented by numbers from 1–4 in the graph for simplification; AC1 (1: 195, 2: 197, 3: 209, 4: 201) and CT2 (1: 188, 2: 184, 3: 196). (D) Discriminant Analysis of Principal Components (DAPC) on microsatellite allele frequencies showing two clear genetic clusters with minimal overlap; colours are as in the upper panel of B.</p

A Tick Gut Protein with Fibronectin III Domains Aids <i>Borrelia burgdorferi</i> Congregation to the Gut during Transmission

<div><p><i>Borrelia burgdorferi</i> transmission to the vertebrate host commences with growth of the spirochete in the tick gut and migration from the gut to the salivary glands. This complex process, involving intimate interactions of the spirochete with the gut epithelium, is pivotal to transmission. We utilized a yeast surface display library of tick gut proteins to perform a global screen for tick gut proteins that might interact with <i>Borrelia</i> membrane proteins. A putative fibronectin type III domain-containing tick gut protein (Ixofin3D) was most frequently identified from this screen and prioritized for further analysis. Immunization against Ixofin3D and RNA interference-mediated reduction in expression of Ixofin3D resulted in decreased spirochete burden in tick salivary glands and in the murine host. Microscopic examination showed decreased aggregation of spirochetes on the gut epithelium concomitant with reduced expression of Ixofin3D. Our observations suggest that the interaction between <i>Borrelia</i> and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a “molecular exit” direction for spirochete egress from the gut.</p></div