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

ABSTRACT: 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.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.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.

SUBMITTER: Hernandez-Castro LE

PROVIDER: S-EPMC5536387 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Hernandez-Castro Luis E LE Paterno Marta M Villacís Anita G AG Andersson Björn B Costales Jaime A JA De Noia Michele M Ocaña-Mayorga Sofía S Yumiseva Cesar A CA Grijalva Mario J MJ Llewellyn Martin S MS

PLoS neglected tropical diseases 20170719 7

<h4>Background</h4>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 suf ...[more]

PMID: 28723901

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Project description:Background2b-RAD (type IIB endonucleases restriction-site associated DNA) approach was invented by Wang in 2012 and proven as a simple and flexible method for genome-wide genotyping. However, there is still plenty of room for improvement for the existent 2b-RAD approach. Firstly, it doesn't include the samples pooling in library preparation as other reduced representation libraries. Secondly, the information of 2b-RAD tags, such as tags numbers and distributions, in most of species are unknown. The purposes of the research are to improve a new 2b-RAD approach which possesses samples pooling, moreover to figure out the characteristic and application potentiality of 2b-RAD tags by bioinformatics analysis.ResultsTwelve adapter1 and an adapter2 were designed. A library approach comprising digestion, ligation, pooling, PCR and size selection were established. For saving costs, we used non-phosphorylated adapters and indexed PCR primers. A F2 population of rice (Oryza sativa .L) was genotyped to validate the new approach. On average, 2000332 high quality reads of each sample were obtained with high evenness. Totally 3598 markers containing 3804 SNPs were discovered and the missing rate was 18.9%. A genetic linkage map of 1385 markers was constructed and 92% of the markers' orders in the genetic map were in accordance with the orders in chromosomes. Meanwhile, the bioinformatics simulation in 20 species showed that the BsaXI had the most widespread recognition sites, indicating that 2b-RAD tags had a powerful application potentiality for high density genetic map. Using modified adapters with a fix base in 3'end, 2b-RAD was also fit for QTL studies with low costs.ConclusionsAn improved 2b-RAD genotyping approach was established in this research and named as I2b-RAD. The method was a simple, fast, cost-effective and multiplex sequencing library approach. It could be adjusted by selecting different enzymes and adapters to fit for alternative uses including chromosomes assembly, QTL fine mapping and even natural population analysis.

Project description:Attalea palms provide primary habitat to Rhodnius spp., vectors of Trypanosoma cruzi. Flying from palms, these blood-sucking bugs often invade houses and can infect people directly or via food contamination. Chagas disease (CD) risk may therefore increase when Attalea palms thrive near houses. For example, Attalea dominate many deforested landscapes of eastern Amazonia, where acute-CD outbreaks are disturbingly frequent. Despite this possible link between deforestation and CD risk, the population-level responses of Amazonian Attalea and their resident Rhodnius to anthropogenic landscape disturbance remain largely uncharted. We studied adult Attalea palms in old-growth forest (OGF), young secondary forest (YSF), and cattle pasture (CP) in two localities of eastern Amazonia. We recorded 1856 Attalea along 10 transects (153.6 ha), and detected infestation by Rhodnius spp. in 18 of 280 systematically-sampled palms (33 bugs caught). Distance-sampling models suggest that, relative to OGF, adult Attalea density declined by 70-80% in CP and then recovered in YSF. Site-occupancy models estimate a strong positive effect of deforestation on palm-infestation odds (βCP-infestation = 4.82±1.14 SE), with a moderate decline in recovering YSF (βYSF-infestation = 2.66±1.10 SE). Similarly, N-mixture models suggest that, relative to OGF, mean vector density sharply increased in CP palms (βCP-density = 3.20±0.62 SE) and then tapered in YSF (βYSF-density = 1.61±0.76 SE). Together, these results indicate that disturbed landscapes may support between ~2.5 (YSF) and ~5.1 (CP) times more Attalea-dwelling Rhodnius spp. per unit area than OGF. We provide evidence that deforestation may favor palm-dwelling CD vectors in eastern Amazonia. Importantly, our landscape-disturbance effect estimates explicitly take account of (i) imperfect palm and bug detection and (ii) the uncertainties about infestation and vector density arising from sparse bug data. These results suggest that incorporating landscape-disturbance metrics into the spatial stratification of transmission risk could help enhance CD surveillance and prevention in Amazonia.

Project description:BackgroundChagas disease is a serious public health problem in Latin America where about ten million individuals show Trypanosoma cruzi infection. Despite significant success in controlling domiciliated triatomines, sylvatic populations frequently infest houses after insecticide treatment which hampers long term control prospects in vast geographical areas where vectorial transmission is endemic. As a key issue, the spatio-temporal dynamics of sylvatic populations is likely influenced by landscape yet evidence showing this effect is rare. The aim of this work is to examine the role of land cover changes in sylvatic triatomine ecology, based on an exhaustive field survey of pathogens, vectors, hosts, and microhabitat characteristics' dynamics.Methodology and principal findingsThe study was performed in agricultural landscapes of coastal Ecuador as a study model. Over one year, a spatially-randomized sampling design (490 collection points) allowed quantifying triatomine densities in natural, cultivated and domestic habitats. We also assessed infection of the bugs with trypanosomes, documented their microhabitats and potential hosts, and recorded changes in landscape characteristics. In total we collected 886 individuals, mainly represented by nymphal stages of one triatomine species Rhodnius ecuadoriensis. As main results, we found that 1) sylvatic triatomines had very high T. cruzi infection rates (71%) and 2) densities of T. cruzi-infected sylvatic triatomines varied predictably over time due to changes in land cover and occurrence of associated rodent hosts.ConclusionWe propose a framework for identifying the factors affecting the yearly distribution of sylvatic T. cruzi vectors. Beyond providing key basic information for the control of human habitat colonization by sylvatic vector populations, our framework highlights the importance of both environmental and sociological factors in shaping the spatio-temporal population dynamics of triatomines. A better understanding of the dynamics of such socio-ecological systems is a crucial, yet poorly considered, issue for the long-term control of Chagas disease.

Project description:Gilthead sea bream (Sparus aurata) is a species of paramount importance to the Mediterranean aquaculture industry, with an annual production exceeding 140,000 metric tonnes. Pasteurellosis due to the Gram-negative bacterium Photobacterium damselae subsp. piscicida (Phdp) causes significant mortality, especially during larval and juvenile stages, and poses a serious threat to bream production. Selective breeding for improved resistance to pasteurellosis is a promising avenue for disease control, and the use of genetic markers to predict breeding values can improve the accuracy of selection, and allow accurate calculation of estimated breeding values (EBV) of non-challenged animals. In the current study, a population of 825 sea bream juveniles, originating from a factorial cross between 67 broodfish (32 sires; 35 dams), were challenged by 30 min immersion with 1 × 10(5) CFU virulent Phdp. Mortalities and survivors were recorded and sampled for genotyping by sequencing. The 2b-RAD sequencing approach was used to generate genome-wide SNP genotypes for all samples. A high-density linkage map containing 12,085 SNPs grouped into 24 linkage groups (consistent with the karyotype) was constructed. The heritability of surviving days (censored data) was 0.22 (95% highest density interval: 0.11-0.36) and 0.28 (95% highest density interval: 0.17-0.4) using the pedigree and the genomic relationship matrix respectively. A genome wide association study did not reveal individual SNPs significantly associated with resistance at a genome wide significance level. Genomic prediction approaches were tested to investigate the potential of the SNPs obtained by 2bRAD for estimating breeding values for resistance. The accuracy of the genomic prediction models (r = 0.38 - 0.46) outperformed the traditional BLUP approach based on pedigree records (r = 0.30). Overall results suggest that major quantitative trait loci (QTL) affecting resistance to pasteurellosis were not present in this population, but highlight the effectiveness of 2b-RAD genotyping by sequencing for genomic selection in a mass spawning fish species.

Project description:SIFamides are a family of highly conserved neuropeptides in arthropods, and in insects are mainly expressed in four medial neurons in the pars intercerebralis of the brain. Although SIFamide has been shown to influence sexual behavior, feeding, and sleep regulation in holometabolous insects such as Drosophila melanogaster, little is known about its role in hemimetabolous insects, including the blood-sucking bug, Rhodnius prolixus. In this study, we confirm the nucleotide sequence for R. prolixus SIFamide (Rhopr-SIFa) and find characteristic phenotypic expression of SIFamide in four cells of the pars intercerebralis in the brain. In addition to extensive SIFa projections throughout the entire central nervous system, SIFamidergic processes also enter into the corpus cardiacum, and project along the dorsal vessel, suggestive of Rhopr-SIFa acting as a neurohormone. Physiologically, Rhopr-SIFamide induces dose-dependent increases in heartbeat frequency in vitro suggesting the presence of peripheral receptors, and thereby indicating Rhopr-SIFa is released to act upon peripheral targets. We also explore the function of Rhopr-SIFa in R. prolixus, specifically in relation to feeding, since R. prolixus is a blood-gorging insect and a vector for Chagas disease. The intensity of SIFamide-like staining in the neurons in the brain is diminished 2 h following feeding, and restocking of those cells is finished 24 h later, indicating Rhopr-SIFa may be released at feeding. The results of temporal qPCR analysis were consistent with the immunohistochemical findings, showing an increase in Rhopr-SIFa transcript expression in the brain 2 h after feeding. We also observed enhanced feeding (size of meal) in insects injected with Rhopr-SIFa whereas insects with RNAi-mediated knockdown of the Rhopr-SIFa transcript consumed a significantly smaller blood meal relative to controls. These data suggest that the four SIFamidergic neurons and associated arborizations may play an important function in the neuronal circuitry controlling R. prolixus feeding, with Rhopr-SIFa acting as a central and peripheral neuromodulator/neurohormone.

Project description:Effective control of Chagas disease vector populations requires a good understanding of the epidemiological components, including a reliable analysis of the genetic structure of vector populations. Rhodnius ecuadoriensis is the most widespread vector of Chagas disease in Ecuador, occupying domestic, peridomestic and sylvatic habitats. It is widely distributed in the central coast and southern highlands regions of Ecuador, two very different regions in terms of bio-geographical characteristics. To evaluate the genetic relationship among R. ecuadoriensis populations in these two regions, we analyzed genetic variability at two microsatellite loci for 326 specimens (n=122 in Manabí and n=204 in Loja) and the mitochondrial cytochrome b gene (Cyt b) sequences for 174 individuals collected in the two provinces (n=73 and=101 in Manabí and Loja respectively). The individual samples were grouped in populations according to their community of origin. A few populations presented positive FIS, possible due to Wahlund effect. Significant pairwise differentiation was detected between populations within each province for both genetic markers, and the isolation by distance model was significant for these populations. Microsatellite markers showed significant genetic differentiation between the populations of the two provinces. The partial sequences of the Cyt b gene (578bp) identified a total of 34 haplotypes among 174 specimens sequenced, which translated into high haplotype diversity (Hd=0.929). The haplotype distribution differed among provinces (significant Fisher's exact test). Overall, the genetic differentiation of R. ecuadoriensis between provinces detected in this study is consistent with the biological and phenotypic differences previously observed between Manabí and Loja populations. The current phylogenetic analysis evidenced the monophyly of the populations of R. ecuadoriensis within the R. pallescens species complex; R. pallescens and R. colombiensis were more closely related than they were to R. ecuadoriensis.

Dataset Information

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

Publications

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

Similar Datasets

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