Triatoma infestans is the main vector of Trypanosoma cruzi, the protozoan agent of Chagas’ disease, in South America between latitudes 10°S and 46°S. The genetic analysis of vector populations may be useful in entomologic surveillance of Chagas’ disease vector control programs because it may provide information on the source of the insects in recolonized areas after insecticide treatment. Previous analyses of 412-basepair5 (bp) segments of the mitochondrial cytochrome B gene did not show variation within T. infestans populations.1 Here, we analyzed mitochondrial DNA (mtDNA) fragments of the 12S and 16S ribosomal RNA genes in 40 specimens from natural populations of T. infestans in localities in four provinces of Argentina (Table 1). All sites were subjected to the same sampling strategy. The insects were collected in each place from different houses or peridomiciliary sites. Fragments of the 12S (371 bp) and 16S (507 bp) genes were sequenced from asymmetric polymerase chain reaction products by manual and automatic sequencing, respectively.2–4 DNA sequences were aligned using the CLUSTAL method implemented in MegAlign, version 1.01 (LaserGene; DNASTAR, Madison, WI). Pairwise FST estimations, a measure of the variance of gene frequencies between populations, and the analysis of molecular variance were carried out using the software package ARLEQUIN 1.1.5–7
The DNA sequence comparisons of the 12S and 16S (878 bp) genes revealed 13 haplotypes determined by 17 variable sites (Table 2) (GenBank accession numbers AY226891-AY226906). Haplotype A is present in all the localities and only the geographically closest localities of El Jardín and Chancaní shared haplotypes C and A/C. The other 10 haplotypes were present exclusively in one of the populations. These private haplotypes, found in the majority of the populations, suggest limited current levels of genetic exchange. The haplotype network (Figure 1) obtained with the TCS software indicates that the different sequence types observed would have derived from a common ancestral haplotype (A), which persists in the current populations of T. infestans after the process of dispersion of this species.8
Intra-individual variation was observed in six of the insects analyzed (Table 2). Each heteroplasmic haplotype was entered into the programs and used in the calculations of variability and FST as two individual different sequences. The percentage of variation within populations was 94.4% and only 5.6% among populations. The levels of genetic differentiation between the populations of Chancaní and Santa María, and Chancaní and Las Lomitas were significant (FST = 0.165, P < 0.01 and 0.173, P < 0,05, respectively), suggesting that the magnitude of gene flow between these populations is not sufficiently large to mask differences eventually produced by genetic drift or local selective pressures.
El Jardín, with seven haplotypes, showed the highest haplotypic diversity, and Chancaní, with five haplotypes, showed the highest degree of nucleotide diversity (Table 2).9 The effects of random genetic drift are more likely to cause losses in genetic variation when the insecticide treatments produce severe population reductions (bottlenecks). The haplotypic diversity and the private haplotypes found in the treated area of El Jardín and Chancaní indicate that the reduction in the population size did not avoid the recovery of the populations apparently from survivors of the same area. In Las Lomitas, where A is the most frequent haplotype, the presence of three private haplotypes indicates an intermediate reduction of genetic variation. Conversely, although the small sample size for Santa Rosa may have limited the finding of variability, the absence of variation in this locality, as well as the low variability observed in Santa Maria, suggest severe bottlenecks derived from the insecticide treatments.
Collection sites of Triatoma infestans and number of individuals studied in Argentina
|Site of collection||Latitude||Longitude||Sample size|
|Santa María. Catamarca||26° 41′ S||66° 02′ W||8|
|Santa Rosa, Valle Viejo, Catamarca||28° 28′ S||65° 47′ W||3|
|El Jardín, General San Martín, La Rioja||31° 26′ S||65° 58′ W||9|
|Chancaní, Pocho, Co′rdoba||31° 26′ S||65° 28′ W||10|
|Las Lomitas, Patiño, Formosa||24° 42′ S||60° 35′ W||10|
Haplotypes, haplotype diversity, and nucleotide diversity found in the 12S and 16S ribosomal RNA genes in populations of Triatoma infestans in Argentina*
|9 2||1 2 2||1 2 8||2 4 4||3 6 3||1 0||1 5||1 8||1 4 5||1 5 7||1 8 9||2 7 4||3 0 7||3 2 2||4 0 4||4 0 5||4 9 6|
|* 1 = Santa María, Catamarca; 2 = Santa Rosa, Valle Viejo, Catamarca; 3 = El Jardín, General San Martín, La Rioja; 4 = Chancaní, Pocho, Córdoba; 5 = Las Lomitas, Patiño, Formosa.|
|Variable nucleotide positions are indicated in sequential order in both genes. The sequence of the haplotype A is used as a reference. Deletions relative to other sequences are denoted by a dash. Nucleotides identical to the reference are indicated by a dot. Intra-individual variation is indicated as r (a/g), y (t/c), and w (a/t).|
|Number of haplotypes||2||1||7||7||4|
|Haplotype diversity (h = 1 − ∑fi2)||0.219||0.000||0.840||0.740||0.480|
|Nucleotide diversity (π, percent)||0.029||0.000||0.123||0.219||0.083|
Authors’ addresses: Beatriz A. García and Candela Manfredi, Cátedra de Bioquímica y Biología Molecular, Pabellón Argentina 2° Piso, Ciudad Universitaria, 5016 Córdoba, Argentina, Telephone: 54-351-433-3024, Fax: 54-351-433-3072 E-mail:
Acknowledgment: We thank Octavio Fusco (Servicio de Secuenciación del Instituto Nacional de Parasitología Dr. Mario Fatala Chabén) for sequencing the 16S ribosomal DNA sequences.
Financial support: This research was partially supported by the grants from the Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba and Banco de la Nación Argentina, the Secretaría de Ciencia y Tecnología de la Universidad Nacional de La Rioja, and Mundo Sano. Beatriz A. Garcia, Laura Fichera, and Elsa L. Segura are Career Investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina.
Monteiro FA, Pérez R, Panzera F, Dujardin JP, Galvão C, Rocha D, Noireau F, Schofield C, Beard CB, 1999. Mitochondrial DNA variation of Triatoma infestans populations and its implication on the specific status of T. melanosoma.Mem lnst Oswaldo Cruz Rio J 94 :229–238.
García BA, 1999. Molecular phylogenetic relationships among species of the genus Triatoma. Carcavallo RU, Galíndez Girón I, Jurberg J, Lent H, eds. Atlas of Chagas’ Disease Vectors in the Americas. Volume III. Rio de Janeiro: Editora Fiocruz, 971–980.
García BA, Powell JR, 1998. Phylogeny of species of Triatoma (Hemiptera: Reduviidae) based on mitochondrial DNA sequences. J Med Entomol 35 :232–238.
García BA, Moriyama EN, Powell JR, 2001. Mitochondrial DNA sequences of triatomines (Hemiptera: Reduviidae): phylogenetic relationships. J Med Entomol 38 :675–683.
Excoffier L, Smouse PE, Quattro JM, 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131 :479–491.
Schneider S, Kueffer JM, Roessli D, Excoffier L, 1997. Arlequin, Version 1.1: A Software for Population Genetic Data Analysis. Geneva, Switzerland: Genetics and Biometry Laboratory, University of Geneva.
Nei M, 1987. Molecular Evolutionary Genetics. New York: Columbia University Press.