Additional file 1: of Aedes aegypti in the Black Sea: recent introduction or ancient remnant?

Table S1. Population information for the Ae. aegypti samples used in this study. Populations represented in both microsatellite and SNP datasets are indicated by bold characters. Table S2. Priors and posteriors for the ABC analysis testing scenarios on the origin of Black Sea populations. Table S3. Climatic data for Black Sea sampling localities (indicated by bold characters) and indicative worldwide sampling localities where Ae. aegypti is established as downloaded from www.worldclim.com . Table S4. Genetic diversity. Summary of the population genetic diversity statistics for the 56 populations of Ae. aegypti consisting the microsatellite dataset. Figure S1. STRUCTURE bar plots based on the microsatellite dataset, including equal number of populations from each region. Population names are reported on their X axes. For each STRUCTURE run only the number of genetic clusters supported by the Evanno method is presented. For details see legend of Fig. 4. Figure S2. STRUCTURE bar plots based on the microsatellite dataset, including equal number of Ae. ae. aegypti populations from each region. For each STRUCTURE run only the number of genetic clusters supported by the Evanno method is presented. For details see legend of Fig. 4. Figure S3. Principal Components Analysis (PCA) on the global (a) and the Ae. ae. aegypti (b) microsatellite dataset as implemented and plotted using the ade4 package in R. Figure S4. Discriminant Analysis of Principal Components (DAPC) for the Ae. ae. aegypti populations based on the microsatellite dataset. Figure S5. Discriminant Analysis of Principal Components (DAPC) for the Ae. aegypti populations collected from Turkey and Georgia based on the microsatellite dataset. Figure S6. Assignment test as implemented in Geneclass2 for the Black Sea populations and using the remaining worldwide populations as reference panel. (PDF 8863 kb

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

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

Intrathoracic injection of the Ho Chi Minh (HCM), Patilas (PAT) and Orlando (ORL) strains of <i>Ae</i>. <i>aegypti</i> with ZIKV results in transmission to mice.

<p>HCM, PAT and ORL strains of <i>Ae</i>. <i>aegypti</i> were infected by intrathoracic injection with 10³ PFU of ZIKV, and after 7 or 10 days allowed to feed on naïve mice. (A) Experimental workflow for the ZIKV injection experiments. (B) Blood was collected every other day for one week from naïve mice fed on by mosquitoes intrathoracically-infected with ZIKV for 7 or 10 days, and analyzed for ZIKV infection by qRT-PCR. ZIKV RNA levels were normalized to mouse <i>β</i> actin (ACTB) RNA levels. (C) Naïve mice fed on by mosquitoes intrathoracically-infected with ZIKV were monitored for survival for at least 32 days after infected mosquitoes-feeding. Data shown are pooled from at least two independent experiments. (n = 6/group for ORL-Day 7; n = 7/group for ORL-Day 10; n = 7/group for PAT-Day 7; n = 7/group for PAT-Day 10; n = 7/group for HCM-Day 7; n = 6/group for HCM-Day 10.) Significance was calculated using two-way ANOVA with a post-hoc Tukey test. ** P<0.01.</p

Origin of the Dengue Fever Mosquito, <i>Aedes aegypti</i>, in California

<div><p>Dengue fever is among the most widespread vector-borne infectious diseases. The primary vector of dengue is the <i>Aedes aegypti</i> mosquito. <i>Ae. aegypti</i> is prevalent in the tropics and sub-tropics and is closely associated with human habitats outside its native range of Africa. While long established in the southeastern United States of America where dengue is re-emerging, breeding populations have never been reported from California until the summer of 2013. Using 12 highly variable microsatellite loci and a database of reference populations, we have determined that the likely source of the California introduction is the southeastern United States, ruling out introductions from abroad, from the geographically closer Arizona or northern Mexico populations, or an accidental release from a research laboratory. The power to identify the origin of new introductions of invasive vectors of human disease relies heavily on the availability of a panel of reference populations. Our work demonstrates the importance of generating extensive reference databases of genetically fingerprinted human-disease vector populations to aid public health efforts to prevent the introduction and spread of vector-borne diseases.</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

Transmission of ZIKV to naïve mice in Ho Chi Minh (HCM), Patilas (PAT) and Orlando (ORL) strains of <i>Ae</i>. <i>aegypti</i>, after taking a blood meal on mice.

<p>HCM, PAT and ORL strains of <i>Ae</i>. <i>aegypti</i> were infected with ZIKV by allowing mosquitoes to ingest a blood meal from mice infected with ZIKV. (A) Experimental workflow for the ZIKV oral infection experiments. (B) Blood was collected every other day for one week from naïve mice fed on by mosquitoes infected with ZIKV for 7 or 10 days, and analyzed for ZIKV infection by qRT-PCR. ZIKV RNA levels were normalized to mouse <i>β</i> actin (ACTB) RNA levels. (C) Naïve mice fed on by mosquitoes orally-infected with ZIKV were monitored for survival for at least 30 days after infected mosquitoes-feeding. Data shown are pooled from at least two independent experiments. (n = 8/group for ORL-Day 7; n = 8/group for ORL-Day 10; n = 5/group for PAT-Day 7; n = 5/group for PAT-Day 10; n = 7/group for HCM-Day 7; n = 5/group for HCM-Day 10.) Each data point represents an individual mouse. Significance was calculated using two-way ANOVA with a post-hoc Tukey test. * P<0.05.</p

Genetic structure within pantropical populations of <i>Aedes aegypti</i>.

<p>STRUCTURE bar plots indicating relatedness of <i>Aedes aegypti</i> populations based on 12 microsatellite loci. Each vertical bar represents an individual. The height of each bar represents the probability of assignment to each of K optimal clusters (different colors) determined using the Delta K method. (<b>A</b>) North America and Asian populations (K = 2), and (<b>B</b>) North American populations (K = 3). (<b>C</b>) Map indicating the North American geographic locations sampled in this study. (<b>Δ</b>) California, (○) other locations in North America.</p

Individual and group mosquito genetic assignments.

<p>Percentage of individuals from Madera (<b>A</b>), Fresno (<b>B</b>), and San Mateo (<b>C</b>) counties assigned with the highest probability to each of the reference populations. (<b>D</b>) Scores calculated for each of the reference populations after group assignment of each of three California populations. Assignments were performed using Bayesian criteria for likelihood estimation with GENECLASS 2.0.</p

Genetic diversity of <i>Aedes aegypti</i> populations.

<p>H_o = observed heterozygosity; H_e = expected heterozygosity; AR = Allelic richness estimated by rarefaction (N = 30 genes).</p><p>*Pantropical = mean across populations from Asia and the Americas.</p><p>**Lab strains = mean across Hamburg, Rockefeller, and Liverpool laboratory strains provided by David Severson (University of Notre Dame, Indiana).</p