Beatriz A. García

Microsatellite analysis of genetic structure in natural Triatoma infestans (Hemiptera: Reduviidae) populations from Argentina: its implication in assessing the effectiveness of Chagas’ disease vector control programmes

The genetic structure in populations of the Chagas’ disease vector Triatoma infestans was examined. Comparisons of the levels of genetic variability in populations of this species from areas with different periods since last insecticide treatment and from areas that never received treatment were also carried out. A total of 598 insects from 19 populations were typed for 10 polymorphic microsatellite loci. The average observed and expected heterozygosities ranged from 0.186 to 0.625 and from 0.173 to 0.787, respectively. Genetic drift and limited gene flow appear to have generated a substantial degree of genetic differentiation among the populations of T. infestans. Departures from Hardy–Weinberg expectations due to an excess of homozygotes suggested the presence of null alleles and population subdivision. Microgeographical analysis supports the existence of subdivision in T. infestans populations. Levels of genetic diversity in the majority of the populations of T. infestans from insecticide‐treated localities were similar or higher than those detected in populations from areas without treatment. Since the populations of T. infestans are subdivided, a population bottleneck would result in independent genetic drift effects that could randomly preserve different combinations of alleles in each subpopulation. These events followed by a rapid population growth could have preserved high levels of genetic diversity. This study supports the hypothesis of vector population recovery from survivors of the insecticide‐treated areas and therefore highlights the value of population genetic analyses in assessing the effectiveness of Chagas’ disease vector control programmes.

Phylogeny of Triatomine Vectors of Trypanosoma cruzi Suggested by Mitochondrial DNA Sequences

The subfamily Triatominae (Hemiptera: Reduviidae) comprises hematophagous insects, most of which are actual or potential vectors of Trypanosoma cruzi, the protozoan agent of Chagas’ disease (American trypanosomiasis). DNA sequence comparisons of mitochondrial DNA (mtDNA) genes were used to infer phylogenetic relationships among 32 species of the subfamily Triatominae, 26 belonging to the genus Triatoma and six species of different genera. We analyzed mtDNA fragments of the 12S and 16S ribosomal RNA genes (totaling 848–851 bp) from each of the 32 species, as well as of the cytochrome oxidase I (COI, 1447 bp) gene from nine. The phylogenetic analyses unambiguously supported several clusters within the genus Triatoma. In the morphological classification, T. costalimai was placed tentatively within the infestans complex while T. guazu was not included in any Triatoma complex. The placement of these species in the molecular phylogeny indicated that both belong to the infestans complex. We confirmed with a strong support the inclusion of T. circummaculata, a member of a different complex based on morphology, within the infestans complex. On the other hand, the present phylogenetics analysis did not support the monophyly of the infestans complex species as it was suggested in our previous studies. While no strong inference of polyphyly of the genus Triatoma was provided by the bootstrap analyses, the other species belonging to Triatomini analyzed could not be distinguished from the species of Triatoma.

Mitochondrial DNA Sequences of Triatomines (Hemiptera: Reduviidae): Phylogenetic Relationships

Abstract The phylogenetic relationships among 18 species of Triatominae were inferred based on mitochondrial DNA (mtDNA) sequences. The species of Triatoma included 11 belonging to the infestans complex [T. infestans (Klug), T. guasayana Wygodzinsky & Abalos, T. sordida (Stål), T. platensis Neiva, T. brasiliensis Neiva, T. rubrovaria (Blanchard), T. vitticeps (Stål), T. delpontei Romaña & Abalos, T. maculata (Erichson), T. patagonica Del Ponte, and T. matogrossensis Leite & Barbosa] and four others of the same genus but of different complexes [T. circummaculata (Stål), T. protracta (Uhler), T. dimidiata (Latreille), and T. mazzottii Usinger]. As possible outgroups we used Mepraia spinolai Mazza, Panstrongylus megistus (Burmeister), and Rhodnius prolixus Stål. We analyzed mtDNA fragments of the 12S and 16S ribosomal RNA genes from each of the 18 species, as well as of the cytochrome oxidase I (COI) gene from nine. The 12S, 16S, and COI gene sequences were analyzed individually and combined. All of the phylogenetic analyses unambiguously supported two clusters: one including T. infestans, T. platensis, and T. delpontei, and the other T. sordida and T. matogrossensis. Inclusion of T. circummaculata into the infestans complex was confirmed, although this is in disagreement with the morphological classification. On the other hand, our analyses showed that T. dimidiata is closely related to a phyllosoma complex species, T. mazzottii. This is consistent with the tentative classification previously made based on morphological characters. The issue of the monophyly of the genus Triatoma remains unresolved.

Genotypes and clinical aspects associated with bone mineral density in Argentine postmenopausal women

The aim of this study was to determine genotypes and clinical aspects associated with bone mineral density (BMD) in postmenopausal women from Córdoba, Argentina. Polymorphisms were assessed by RFLP-PCR technique using BsmI and FokI for vitamin D receptor gene (VDR) and XbaI and PvuII for estrogen receptor-α gene (ERα) as restrictases. Sixty-eight healthy, 54 osteopenic, and 64 osteoporotic postmenopausal women were recruited. Femoral neck and lumbar spine BMD were inversely correlated with age in the entire analyzed population. Height was lower in osteopenic and osteoporotic women as compared to healthy women (P < 0.05). Weight and body mass index (BMI) were the lowest in osteoporotic women (P < 0.01 versus healthy group). Serum procollagen type I Nterminal propeptide (PINP) was higher in osteoporotic women as compared to the other groups. Distribution of VDR and ERα genotypes was similar in the three groups. Genotype bb (VDR) was associated with low values of lumbar BMD in the healthy group (P < 0.05 versus genotype Bb), and with low values of femoral BMD (P < 0.05 versus genotype BB) in osteoporotic women. BB*Pp interaction was associated with the highest femoral neck BMD (P < 0.05), whereas the bb*xx interaction was associated with the lowest femoral neck BMD in the total population analyzed (P < 0.05). In conclusion, parameters such as age, height, weight, BMI, serum PINP, VDR genotypes, and interactions between VDR and ERá genotypes could be useful to predict a decrease in BMD in Argentine postmenopausal women.

Mitochondrial 16S DNA variation in populations of Triatoma infestans from Argentina.

Abstract Variation in the mtDNA 16S ribosomal RNA gene in populations of Triatoma infestans (Klug) was surveyed. DNA sequence comparisons yielded 18 haplotypes among 130 individuals from 16 localities that represent a large proportion of the range of T. infestans in Argentina. The most common genotype in all populations was found in 76.9% of individuals and two other haplotypes were shared among different populations. The remaining 15 haplotypes were present exclusively in one of the populations, suggesting currently low levels of genetic exchange. Analysis of mtDNA 16S sequences uncovered substantial genetic variation among T. infestans populations. Haplotype and nucleotide diversities varied among populations, from 0% to 0.84% and 0% to 0.29%, respectively. It appears that this locus has a low mutation rate. Uncorrected pairwise differences of T. infestans haplotypes ranged from 0% to 1.2%. The molecular phylogeny supported the monophyly of T. infestans haplotypes and clustered two different pairs of haplotypes with a moderate degree of bootstrap support (∼ 60%). Mitochondrial DNA phylogeographic differentiation was not evident, suggesting a recent rapid spread of the species. Analysis of molecular variance showed hierarchical structure in the data. Considerably less variation was found among T. infestans populations from the northwest and northeast regions than among those belonging to the central area. Such a lack of variation may be indicative of one or more past population bottlenecks.

Protein polymorphism and genetic distance in South American cricetid rodents of the genus Calomys

Allozymic variation in population samples of Calomys laucha, C. musculinus and C. venustus collected simultaneously from the province of Córdoba, Argentina, was analyzed by gel electrophoresis. These three species showed high levels of polymorphism: for C. laucha, the P value was 72.7% and the H value was 0.147 (19 loci); for C. musculinus, P=61.9% and H=0.149 (20 loci); for C. venustus, P=66.7% and H=0.146 (18 loci). These species are non-fossorial generalists, have a high reproductive rate and live in disturbed habitats. The similarity indices (S) range between 0.613 and 0.681. On the basis of genetic distance, D (between 0.354 and 0.476), divergence time was estimated to be from 6.3 to 8.5 million years (late Miocene). Even admitting some imprecision for this estimate, the remarkable differences at the structural gene level shown by the species analyzed, suggest they had an early origin.

Molecular phylogeography of the Chagas' disease vector Triatoma infestans in Argentina

Triatoma infestans is the main vector of Chagas’ disease in South America between latitudes 10°S and 46°S. A multilocus microsatellite data set of 836 individuals from 27 populations of T. infestans, from all its range of distribution in Argentina, was analyzed. Our results favor the hypothesis of two independent migration events of colonization in Argentina and secondary contacts. The majority of the populations of the western provinces of Catamarca, La Rioja, San Juan and the west of Cordoba province, had almost no shared ancestry with the rest of the populations analyzed. Probably those populations, belonging to localities close to the Andean region, could have been established by the dispersal line of T. infestans that would have arrived to Argentina through the Andes, whereas most of the rest of the populations analyzed may have derived from the dispersal line of T. infestans in non-Andean lowlands. Among them, those from the provinces of Formosa, Chaco, Santiago del Estero and Santa Fe shared different percentages of ancestry and presented lower degree of genetic differentiation. The migratory movement linked to regional economies and possibly associated with passive dispersal, would allow a higher genetic exchange among these populations of T. infestans. This study, using microsatellite markers, provides a new approach for evaluating the validity of the different hypotheses concerning the evolutionary history of this species. Two major lineages of T. infestans, an Andean and non-Andean, are suggested.

Identification of three cytochrome P450 genes in the Chagas' disease vector Triatoma infestans: Expression analysis in deltamethrin susceptible and resistant populations

Cytochrome P450 monooxygenases play a predominant role in the metabolism of insecticides. Many insect P450 genes have frequently been associated with detoxification processes allowing the insect to become tolerant or resistant to insecticides. The increases of expression of P450 genes at transcriptional level are often consider responsible for increasing the metabolism of insecticides and seems to be a common phenomenon in the evolution of resistance development in insects. As pyrethroid resistance has been detected in Triatoma infestans, it was of interest to analyze genes associated with resistance to insecticides such as those encoding for cytochromes P450. With this purpose, the cDNA sequences of three cytochrome P450 genes (CYP4EM7, CYP3085B1, and CYP3092A6) were identified in this species. Primers and specific Taqman probes were designed from these sequences to determine their expression by quantitative PCR. The mRNA levels of the cytochrome P450 genes identified were determined from total RNA extracted from pools of fat body collected from individuals of different resistant and susceptible strains of T. infestans, and at different interval times after the topical application of the lethal doses 50% (LD50) of deltamethrin on the ventral abdomen of insects belonging to the different populations analyzed. It was detected overexpression of the CYP4EM7 gene in the most resistant strain of T. infestans and the expression of the three cytochrome P450 genes isolated was induced by deltamethrin in the susceptible and resistant populations included in this study. These results suggest that these genes would be involved in the detoxification of deltamethrin and support the hypothesis that considers to the cytochrome P450 genes of importance in the development of pyrethroid resistance.

TEMPORAL FLUCTUATION OF EFFECTIVE SIZE IN POPULATIONS OF CALOMYS MUSCULINUS (MURIDAE: SIGMODONTINAE)

Abstract Calomys musculinus is the natural reservoir of Junin virus, the etiological agent of Argentine hemorrhagic fever. In this paper we measure the effective size of 2 populations of the rodent over a 2-year period. Twenty enzyme-coding loci were analyzed using vertical starch gel electrophoresis. Effective population sizes (Nes) were estimated by the pseudolikelihood method in 2 populations 280 km apart in central Argentina. Both populations experienced marked seasonal changes in relative density and in Ne (between 19.8 and infinity). Changes in percentage of polymorphic loci and mean number of alleles per locus were statistically significant and were roughly correlated with density and Ne. Observed changes in heterozygosity, in contrast, were not significant. After low-density periods, mixing of surviving individuals coming from different demes may play an important role in the maintenance of variability and recovery of Ne in populations of C. musculinus.

Molecular Population Genetics and Evolution of the Chagas’ Disease Vector Triatoma infestans (Hemiptera: Reduviidae)

Triatoma infestans (Klug) is the main vector of Chagas’ disease in the Southern Cone of Latin America between the latitudes 10° S and 46° S. The long-term effectiveness of the control campaigns is greatly dependent upon the vector population structure. Mitochondrial DNA (mtDNA) genes have been used in a number of T. infestans population genetic analyses. However, the maternally inherited markers as well as nuclear ribosomal DNA analyzed until the present exhibited low or limited levels of variation. Analyses based on microsatellite markers strongly supported the existence of some type of stratification in T. infestans populations and supported the hypothesis of vector population recovery from survivors of the insecticide-treated areas, highlighting the value of population genetic analyses in assessing the effectiveness of Chagas’ disease vector control programmes. Although phylogeographic studies have generally suggested a Bolivian Andean origin of T. infestans, they recovered two reciprocal monophyletic groups of T. infestans and Bolivian populations who were not basal as expected for an ancestral group. In addition, a non-Andean origin could not be excluded by mtDNA genealogies that included sylvatic bugs from Gran Chaco. On the other side, mitochondrial and microsatellite markers supported the hypothesis of two independent migration events of colonization and secondary contacts in southern South America. Since the phylogenetic analyses remain inconclusive, more sequences, not only from mitochondrial genes but also from nuclear genes, need to be examined.