2b-RAD genotyping for population genomic studies of Chagas disease vectors: Rhodnius ecuadoriensis in Ecuador

Background: Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. Methodology/Principal findings: The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. Conclusions/Significance: Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment. Author summary: Understanding Chagas disease vector (triatomine) population dispersal is key for the design of control measures tailored for the epidemiological situation of a particular region. In Ecuador, Rhodnius ecuadoriensis is a cause of concern for Chagas disease transmission, since it is widely distributed from the central coast to southern Ecuador. Here, a genome-wide sequencing (2b-RAD) approach was performed in 20 specimens from four communities from Manabí (central coast) and Loja (southern) provinces of Ecuador, and the effectiveness of three type IIB restriction enzymes was assessed. The findings of this study show that this genotyping methodology is cost effective in R. ecuadoriensis and likely in other triatomine species. In addition, preliminary population genomic analysis results detected a signal of population structure among geographically distinct communities and genetic variability within communities. As such, 2b-RAD shows significant promise as a relatively low-tech solution for determination of vector population genomics, dynamics, and spread

Population genomics and geographic dispersal in Chagas disease vectors: landscape drivers and evidence of possible adaptation to the domestic setting

Accurate prediction of vectors dispersal, as well as identification of adaptations that allow blood-feeding vectors to thrive in built environments, are a basis for effective disease control. Here we adopted a landscape genomics approach to assay gene flow, possible local adaptation, and drivers of population structure in Rhodnius ecuadoriensis, an important vector of Chagas disease. We used a reduced-representation sequencing technique (2b-RADseq) to obtain 2,552 SNP markers across 272 R. ecuadoriensis samples from 25 collection sites in southern Ecuador. Evidence of high and directional gene flow between seven wild and domestic population pairs across our study site indicates insecticide-based control will be hindered by repeated re-infestation of houses from the forest. Preliminary genome scans across multiple population pairs revealed shared outlier loci potentially consistent with local adaptation to the domestic setting, which we mapped to genes involved with embryogenesis and saliva production. Landscape genomic models showed elevation is a key barrier to R. ecuadoriensis dispersal. Together our results shed early light on the genomic adaptation in triatomine vectors and facilitate vector control by predicting that spatially-targeted, proactive interventions would be more efficacious than current, reactive approaches

Distribution of triatomine species in domestic and peridomestic environments in central coastal Ecuador.

Although the central coast of the Ecuador is considered endemic for Chagas disease, few studies have focused on determining the risk of transmission in this region. In this study we describe the triatomine household infestation in Manabí province (Central Coast region), determine the rate of Trypanosoma cruzi infection and study the risk factors associated with infestation by Rhodnius ecuadoriensis.An entomological survey found three triatomine species (Rhodnius ecuadoriensis, Panstrongylus rufotuberculatus and P. howardi) infesting domiciles in 47.4% of the 78 communities visited (total infestation rate of 4.5%). Four percent of domiciles were infested, and nymphs were observed in 77% of those domiciles. The three species were found in altitudes below 500 masl and in all ecological zones except cloud forest. Within the domicile, we found the three species mostly in bedrooms. Rhodnius ecuadoriensis and P. rufotuberculatus were abundant in bird nests, including chicken coops and P. howardi associated with rats in piles of bricks, in the peridomicile. Triatomine infestation was characterized by high rates of colonization, especially in peridomicile. Flagelates infection was detected in only 12% of the samples by microscopy and Trypanosoma cruzi infection in 42% of the examined triatomines by PCR (n = 372). The most important risk factors for house infestation by R. ecuadoriensis were ecological zone (w = 0.99) and presence of chickens (w = 0.96). Determinants of secondary importance were reporting no insecticide applications over the last twelve months (w = 0.86) and dirt floor (w = 0.70). On the other hand, wood as wall material was a protective factor (w = 0.85).According the results, approximately 571,000 people would be at high risk for T. cruzi infection in Manabí province. A multidisciplinary approximation and the adhesion to a periodic integrated vector management (IVM) program are essential to guarantee sustainable preventive and control strategies for Chagas disease in this region

Peridomestic materials and vegetation, found in rural communities of Loja Province.

<p>^a total n varies among each item from 2,078 to 2,097 due to missing values.</p><p>^b Median and Interquartile Range of distance in meters from domicile (IR)</p><p>Peridomestic materials and vegetation, found in rural communities of Loja Province.</p

Livestock found in rural communities of Loja Province.

<p>^a total n varies among each item from 3,015 to 3,032 due to missing values.</p><p>^b Median and Interquartile Range (IR) of animals per domicile</p><p>Livestock found in rural communities of Loja Province.</p

Characteristics of domiciles from 78 rural communities in Manabí province.

<p>Characteristics of domiciles from 78 rural communities in Manabí province.</p

Characteristic of domiciles from 92 communities located in rural areas of Loja Province.

<p>^a total n varies among each item from 2.996 to 3,039 due missing values.</p><p>^b total n varies among each item from 2,099 to 2,204 due to a change on the questionnaire form in 2008 and some missing values.</p><p>^c Inhabitant crowding = more than 3 people per bedroom.</p><p>Characteristic of domiciles from 92 communities located in rural areas of Loja Province.</p

Triatomine infestation in Loja Province, a region of southern Ecuador having several ecological regions.

<p>Location of each community surveyed is marked. The size of the symbol corresponds to the % of houses infested with triatomines in each community. The colors represent the four triatomine species found: Red = <i>R</i>. <i>ecuadoriensis</i>, blue = <i>T</i>. <i>carrioni</i>, green = <i>P</i>. <i>rufotuberculatus</i> and yellow = <i>P</i>. <i>chinai</i>. Classification of ecological regions as per Sierra et al [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004142#pntd.0004142.ref014" target="_blank">14</a>].</p

Important determinants for house infestation by <i>R</i>. <i>ecuadoriensis</i> in Manabí province, 2009–2011.

<p>Important determinants for house infestation by <i>R</i>. <i>ecuadoriensis</i> in Manabí province, 2009–2011.</p

Comparison of PCR and Microscopy for <i>T</i>. <i>cruzi</i> and <i>T</i>. <i>rangeli</i> detection in triatomines.

<p>ME: microscopy examination;</p><p>PCR: Polymerase Chain Reaction</p><p>Comparison of PCR and Microscopy for <i>T</i>. <i>cruzi</i> and <i>T</i>. <i>rangeli</i> detection in triatomines.</p