Microsatellite variation revealed panmictic pattern for Triatoma brasiliensis (Triatominae: Reduviidae) in rural northeastern Brazil: the control measures implications.

BACKGROUND: Triatoma brasiliensis Neiva, 1911 is the main vector of Trypanosoma cruzi in the caatinga of Northeastern Brazil. Despite of its epidemiological relevance, there are few studies on its genetic variability. Using microsatellite markers, we characterized the variability and dynamics of infestation and reinfestation of T. brasiliensis after residual insecticide spraying in five surveys conducted in a well-defined rural area located in the municipality of Tauá, Ceará, between 2009 and 2015. We evaluated: (1) general variability among local of captures; (2) variability along the time analysis (2009, 2010 and 2015); (3) and reinfestation process. RESULTS: On the analysis (1) global and pairwise FST values suggested absence of clusters among the area. AMOVA indicated that total variation is mainly represented by individual differences. Absence of clustering indicates a panmitic unit, with free gene flow. For (2), Pairwise FST indicated alterations in the genetic profile of the triatomines along the time. (3) Analysis of the reinfestation process showed that the domiciliary units investigated had different sources of infestation despite of its proximity. CONCLUSIONS: Observed homogeneity can be explained by the great dispersal capacity of T. brasiliensis, overlapping the different environments. Persistent house infestation in Tauá may be attributed to the occurrence of postspraying residual foci and the invasion of triatomines from their natural habitats.

Dynamics of Panstrongylus megistus infestation, the primary vector of Trypanosoma cruzi in Minas Gerais, Brazil.

Panstrongylus megistus is considered one of the primary species of epidemiological importance for the transmission of Chagas disease in Brazil due to its wide geographical distribution throughout the national territory, good ability to invade and colonize houses, and high rates of natural infection by the Trypanosoma cruzi. The importance of this species in Minas Gerais has been recognized since the 80s. It is responsible for the high prevalence rates of Chagas disease in the west of the state. Studies conducted in the municipality of Jaboticatubas show that P. megistus is still the most captured vector in the region, even after 40 years of uninterrupted actions of the Chagas Disease Control Program in the municipality. Despite the importance of the species, its population dynamics is little studied. Consequently, crucial genetic information such as genetic diversity and gene flow among environments have not been well characterized yet. In this context, this work presents a population genetic analysis at the microgeographic level using microsatellite markers in samples of P. megistus obtained from Jaboticatubas to better understand the infestation dynamics of the primary vector species of T. cruzi in the region. The observed and expected heterozygosity ranged from 0.26 to 0.47 and 0.47 to 0.65, respectively. Most loci presented Hardy-Weinberg disequilibrium due to the excess of homozygotes. The pairwise Fst ranged from 0.05 to 0.35, with the p value significant for all comparisons, indicating the absence of gene flow between them. The values of Fis found ranged from 0.25 to 0.52, all values of p ≤ 0.05, probably due to structured populations, inbreeding, or null alleles. The results suggest an extended stay of this species in the colonized environments, with rare dispersal to other locations. Such results differ from that observed for Triatoma brasiliensis and Triatoma dimidiata, species that present constant movement, even after colonization of the artificial environment, and are similar to the behavior of Triatoma infestans. Thus, the study reinforces the importance of continuous entomological surveillance in the areas of occurrence of P. megistus to avoid the installation and formation of new foci of household infestation in the region.

Identification and characterization of microsatellite markers for population genetic studies of Panstrongylus megistus (Burmeister, 1835) (Triatominae: Reduviidae).

BACKGROUND: Panstrongylus megistus is the most important vector of Chagas disease in Brazil. Studies show that the principal factor hindering the control of triatomines is reinfestation of houses previously treated with insecticides. Studies at the microgeographic level are therefore necessary to better understand these events. However, an efficient molecular marker is not yet available for carrying out such analyses in this species. The aim of the present study was to identify and characterize microsatellite loci for future population genetic studies of P. megistus. METHODS: This study work consisted of five stages: (i) sequencing of genomic DNA; (ii) assembly and selection of contigs containing microsatellites; (iii) validation of amplification and evaluation of polymorphic loci; (iv) standardization of the polymorphic loci; and (v) verification of cross-amplification with other triatomine species. RESULTS: Sequencing of males and females generated 7,908,463 contigs with a total length of 2,043,422,613 bp. A total of 2,043,690 regions with microsatellites in 1,441,091 contigs were obtained, with mononucleotide repeats being the most abundant class. From a panel of 96 loci it was possible to visualize polymorphisms in 64.55% of the loci. Of the 20 loci genotyped, the number of alleles varied from two to nine with an average of 4.9. Cross-amplification with other species of triatomines was observed in 13 of the loci. CONCLUSIONS: Due to the high number of alleles encountered, polymorphism and the capacity to amplify from geographically distant populations, the microsatellites described here show promise for utilization in population genetic studies of P. megistus.

Identificação e caracterização de marcadores microssatélites de Pansatrongylus megistus (Burmeister, 1835) (Triatominae: Reduviidae)

Os triatomineos sao insetos hematofagos vetores do Trypanosoma cruzi, o agente etiologico da doenca de Chagas. Atualmente sao descritas 152 especies de triatomineos sendo 65 autoctones do Brasil, entre essas especies o Panstrongylus megistus e considerado o de maior importancia epidemiologia no Sudeste do Brasil devido a sua ampla distribuicao geografica alta capacidade de colonizacao, alta antropofilia e altos niveis de infeccao natural. A permanencia deste vetor no ambiente artificial (casas e anexos) tem sido um desafio para o controle da doenca de Chagas (DCh), nao existindo nenhuma evidencia da origem dos processos de (re)infestacao dos domicilios, se esta ocorre a partir de focos externos e/ou residuais, colocando sob risco as populacoes rurais em sua area de ocorrencia. A genetica de populacoes e uma importante ferramenta para estimar a dinamica entre habitats, podendo auxiliar na compreensao da ecoepidemiologia da DCh. Estudos ja realizados com outras especies de triatomineos demonstraram que marcadores microssatelites sao capazes de revelar o fluxo genico em escala microgeografica. No entanto tais marcadores ainda nao haviam sido descritos para esta especie vetora. Neste contexto o objetivo deste trabalho foi identificar e padronizar marcadores microssatelites para P. megistus para serem utilizados em analises futuras de genetica de populacoes. O sequenciamento genomico em plataforma Illumina HiSeqX foi utilizado para montagem de reads e posterior busca de microssatelites. Foram identificadas 2.043.690 regioes utilizando o programa MISA, destas, 96 foram selecionadas para a padronizacao da amplificacao, das quais 79 loci amplificaram por PCR utilizando o DNA de seis exemplares de tres localidades distintas (dois insetos de Santana do Riacho/Minas Gerais, dois insetos de Juquia/Sao Paulo, e dois insetos de uma colonia formada por individuos de diferentes localidades de Minas Gerais). Dentre estes, 20 dos 51 loci polimorficos foram utilizados para a determinacao dos alelos por genotipagem em 19 P. megistus provenientes de: Jaboticatubas/MG (15), Santana do Riacho/MG (2) e Juquia/SP (2); e de um exemplar de: Panstrongylus diasi, Panstrongylus lignarius, Triatoma tibiamaculata, e Triatoma sordida. A determinacao dos alelos foi possivel em 19 loci, sendo 16 polimorficos. O numero de alelos por locus variou de 1 a 9 com medida de 4,7. A heterozigozidade observada variou de 0,15 a 0,57 enquanto a esperada foi de 0,14 a 0,83 com media de 0,25 e 0,59 respectivamente. Foi possivel observar a amplificacao em outra especie de triatomineo em 12 loci. Dessa forma, a estrategia utilizada mostrou-se eficicaz para o desenvolvimento de marcadores de microssatelites. A proxima etapa sera avaliar a dinamica populacional de infestacoes do ambiente artificial por esta especie.