Computational protocol: Hidden Sylvatic Foci of the Main Vector of Chagas Disease Triatoma infestans: Threats to the Vector Elimination Campaign?

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Protocol publication

[…] DNA from bugs assigned to T. infestans (based on morphological evidence) was obtained, PCR-amplified, and sequenced for a 661 bp fragment of the mitochondrial genes cytochrome oxidase I (mtCOI) and a 572 bp fragment of the cytochrome B (mtcytB) gene . Sequences from sylvatic T. infestans were compared with Triatoma spp sequences available at Genbank and from previous surveys on the instraspecific variability of T. infestans , –.Sylvatic T. infestans mtCOI plus mtcytB composite haplotypes were compared with previously recorded haplotypes of D/PD T. infestans from the study villages (collected in 2001–2002), from other more distant (40 km) localities within Santiago del Estero Province (Quilumpa, Km 40, La Loma and Invernada Norte, collected in 2003–2004), and from other Argentinean Provinces more than 300 km apart (Salta, La Rioja, Tucumán and Formosa, collected in 2000–2005). A detailed description of the source localities was published elsewhere . Genetic variability was estimated as the mean number of pairwise differences per site (π), Watterson's estimator (θW) and the haplotype diversity (Hd) with DnaSP 5.0 and a statistical parsimony haplotype network was built with TCS 1.21 .For higher resolution of the relationships between sylvatic and D/PD populations of T. infestans, the multilocus (ML) genotype for 10 microsatellite loci was obtained for sylvatic T. infestans using primers and PCR conditions previously described . ML genotypes were compared with those from T. infestans captured in D/PD sites from Amamá and neighboring villages in October 2002 and April 2004 before full-coverage insecticide spraying . Inter-individual genetic distance based on the complement of the proportion of shared alleles was estimated with MICROSAT 1.5d (, and a neighbor-joining (NJ) tree was built with MEGA 3.0 .Using the genotypes of local D/PD T. infestans as reference populations, we applied the Bayesian based assignment-exclusion test implemented in GENECLASS 2 to individually assign sylvatic individuals to the local pre-spraying D/PD populations (defined as the total gene pool at a given community in each capture date). No post-spraying reference groups could be formed because after community-wide insecticide spraying (2004–2006) most bug collections contained one or a few insects per site that were sparsely distributed throughout the communities (i.e., no established populations of T. infestans were detected). Reference populations were not excluded as the putative origin of the sylvatic insects when the marginal probability exceeded 0.05. We used 100,000 replications and a simulation algorithm .Sibship of T. infestans bugs collected in traps with more than one individual (TN-92 and TN-139) was inferred with the maximum likelihood approach implemented in COLONY 2.0 performing two independent runs and assuming a probability of null alleles of 0.05 in loci ms42, ms64 and ms65 due to departures from Hardy-Weinberg expectations. […]

Pipeline specifications

Software tools DnaSP, MEGA, GeneClass
Application Population genetic analysis
Diseases Tick Infestations, Chagas Disease, Leukemia, T-Cell