Prevalence and distribution of parasites and pathogens of Triatominae from Argentina, with emphasis on Triatoma infestans and Triatoma virus TrV

Chagas’ disease is the most important endemic arthropod-zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas’ disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n = 1646), Triatoma guasayana (n = 4), Triatoma platensis (n = 1) and Triatoma sordida (n = 5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas’ disease regions of Argentina.     

Triatoma patagonica (Hemiptera,Reduviidae), a new host for Triatoma virus

Previous authors demonstrated that Triatoma virus (TrV) is able to infect several species of triatomines when injected with viral inoculum obtained from its original host, T. infestans. Both vertical (transovarian) and horizontal (faecal-oral) mechanisms of viral transmission were also described. In this paper we report the experimental TrV infection of a wild species from southern Argentina, T. patagonica. The inoculum consisted of clarified gut contents of infected T. infestans rubbed on the chicken skin whereupon T. patagonica individuals were fed. The results demonstrate that this is another potential host for the virus, and that the oral route is also effective for experimental interspecific infections.     

Distribution and Evolution of Repeated Sequences in Genomes of Triatominae (Hemiptera-Reduviidae) Inferred from Genomic In Situ Hybridization

The subfamily Triatominae, vectors of Chagas disease, comprises 140 species characterized by a highly homogeneous chromosome number. We analyzed the chromosomal distribution and evolution of repeated sequences in Triatominae genomes by Genomic in situ Hybridization using Triatoma delpontei and Triatoma infestans genomic DNAs as probes. Hybridizations were performed on their own chromosomes and on nine species included in six genera from the two main tribes: Triatomini and Rhodniini. Genomic probes clearly generate two different hybridization patterns, dispersed or accumulated in specific regions or chromosomes. The three used probes generate the same hybridization pattern in each species. However, these patterns are species-specific. In closely related species, the probes strongly hybridized in the autosomal heterochromatic regions, resembling C-banding and DAPI patterns. However, in more distant species these co-localizations are not observed. The heterochromatic Y chromosome is constituted by highly repeated sequences, which is conserved among 10 species of Triatomini tribe suggesting be an ancestral character for this group. However, the Y chromosome in Rhodniini tribe is markedly different, supporting the early evolutionary dichotomy between both tribes. In some species, sex chromosomes and autosomes shared repeated sequences, suggesting meiotic chromatin exchanges among these heterologous chromosomes. Our GISH analyses enabled us to acquire not only reliable information about autosomal repeated sequences distribution but also an insight into sex chromosome evolution in Triatominae. Furthermore, the differentiation obtained by GISH might be a valuable marker to establish phylogenetic relationships and to test the controversial origin of the Triatominae subfamily.     

The ITS-2 of the nuclear rDNA as a molecular marker for populations, species, and phylogenetic relationships in Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease

The nucleotide sequences of the rDNA second internal transcribed spacer (ITS-2) of 31 populations of 12 and 3 species of the two main Triatominae tribes Triatomini and Rhodniini, including the most important Chagas disease vectors, were obtained. Sequence comparisons and parsimony, distance, and maximum-likelihood analyses indicate that ITS-2 is a useful marker for resolving supraspecific, specific, subspecific, and even sometimes population-level relationships in Triatominae. Results were markedly different between species of Triatomini and Rhodniini, suggesting polyphyly. Phylogenetic trees support an old divergence between South American and North-Central American Triatomini and query the validity of some genera (Dipetalogaster, Psammolestes). The very low sequence variation between species of the phyllosoma complex suggests that subspecific ranking would be more appropriate. Triatoma dimidiata proves to be a clearly differentiated species, with several populations evidencing a clinal variation along a north-south axis and a population from Yucatan showing differences consistent with specific status.     

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.     

Genetic variation, population structure, and phylogenetic relationships of Triatoma rubida and T. recurva (Hemiptera: Reduviidae: Triatominae) from the Sonoran Desert, insect vectors of the Chagas' disease parasite Trypanosoma cruzi

Nucleotide and amino acid sequence data from the mitochondrial cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) gene segments were used to gain insights into the population biology and phylogenetic relationships of two species of hematophagous kissing bugs (Hemiptera: Reduviidae: Triatominae) from the Sonoran Desert of northwestern Mexico and southern Arizona, USA, Triatoma rubida (Uhler, 1894) and T. recurva (St?l, 1868), both of which are vectors of the protozoan parasite Trypanosoma cruzi responsible for Chagas' disease. Analysis of molecular variance of gene sequences indicated significant structure among populations of both species from widely separated geographic localities. Phylogenetic analyses of gene and amino acid sequences employing both Bayesian and parsimony methods showed that T. recurva clustered within the phyllosoma complex of Triatoma species from central and southern Mexico with high statistical support, and that it was closely related to T. longipennis. Triatoma dimidiata also was shown to be closely related to the phyllosoma complex, as was T. sanguisuga which has historically been assigned to the lecticularia complex. Analyses of gene sequences were unable to confidently resolve relationships of T. rubida, although weak support for a T. nitida+T. rubida clade was seen under certain conditions. A provisional calibration of a mitochondrial DNA molecular clock for T. rubida, based on geological dates for the vicariant separation of the Baja California peninsula from mainland Mexico, suggested that pairwise sequence divergences for the Cytb and COI genes were 1.1-1.8% and 0.6-1.0% per million years, respectively. Two highly supported sympatric lineages of T. rubida uhleri from southern Arizona, which are hypothesized to have diverged approximately 550,000-900,000 years ago, were detected in the Cytb gene trees.     

Atlas of Mexican Triatominae (Reduviidae: Hemiptera) and vector transmission of Chagas disease

Chagas disease is one of the most important yet neglected parasitic diseases in Mexico and is transmitted by Triatominae. Nineteen of the 31 Mexican triatomine species have been consistently found to invade human houses and all have been found to be naturally infected with Trypanosoma cruzi. The present paper aims to produce a state-of-knowledge atlas of Mexican triatomines and analyse their geographic associations with T. cruzi, human demographics and landscape modification. Ecological niche models (ENMs) were constructed for the 19 species with more than 10 records in North America, as well as for T. cruzi. The 2010 Mexican national census and the 2007 National Forestry Inventory were used to analyse overlap patterns with ENMs. Niche breadth was greatest in species from the semiarid Nearctic Region, whereas species richness was associated with topographic heterogeneity in the Neotropical Region, particularly along the Pacific Coast. Three species, Triatoma longipennis, Triatoma mexicana and Triatoma barberi, overlapped with the greatest numbers of human communities, but these communities had the lowest rural/urban population ratios. Triatomine vectors have urbanised in most regions, demonstrating a high tolerance to human-modified habitats and broadened historical ranges, exposing more than 88% of the Mexican population and leaving few areas in Mexico without the potential for T. cruzi transmission.     

First report of colonies of sylvatic Triatoma infestans (Hemiptera: Reduviidae) in the Paraguayan Chaco, using a trained dog

In the Gran Chaco region, control of Triatoma infestans has been limited by persistent domestic infestations despite the efforts of the Vector Control Services. In Paraguay, this region is the highest endemic area in the country, showing high levels of indoor and outdoor infestation. Although sylvatic T. infestans have been found in the Bolivian and Argentine Chaco, similar searches for sylvatic populations of this species in Paraguay had been unsuccessful over the last 20 years. Here we present a new approach to detecting sylvatic Triatominae, using a trained dog, which has successfully confirmed sylvatic populations of T. infestans and other triatomine species in Paraguay. A total of 22 specimens corresponding to dark morph forms of T. infestans were collected, and 14 were confirmed as T. infestans by the mitochondrial cytochrome B gene analysis. Through this analysis, one of which were previously reported and a second that was a new haplotype. Triatomines were captured from amongst vegetation such as dry branches and hollows trees of different species such Aspidosperma quebracho-blanco, Bulnesia sarmientoi and Stetsonia coryne. The colonies found have been small and without apparent infection with Trypanosoma cruzi. During the study, Triatoma sordida and Triatoma guasayana have also been found in ecotopes close to those of T. infestans.     

Competitive displacement in Triatominae: the Triatoma infestans success

Brazil has just been certificated by Pan American Health Organization as 'free of Chagas disease transmission due to Triatoma infestans'. During the early 1980s, this species of blood-sucking bug alone was considered responsible for approximately 80% of Chagas disease transmission. But it was not always so. The species originally abundant in houses of central and eastern Brazil was Panstrongylus megistus, which seems to have been progressively displaced from houses by T. infestans during the past century. Indeed, T. infestans seems able to displace other Triatominae in artificial environments. Recent studies suggest that it might simply be because T. infestans feeds more efficiently than its Triatominae competitors.     

Trapping Triatominae in silvatic habitats

Large-scale trials of a trapping system designed to collect silvatic Triatominae are reported. Live-baited adhesive traps were tested in various ecosystems and different triatomine habitats (arboreal and terrestrial). The trials were always successful, with a rate of positive habitats generally over 20% and reaching 48.4% for palm trees of the Amazon basin. Eleven species of Triatominae belonging to the three genera of public health importance (Triatoma, Rhodnius and Panstrongylus) were captured. This trapping system provides an effective way to detect the presence of triatomines in terrestrial and arboreal silvatic habitats and represents a promising tool for ecological studies. Various lines of research are contemplated to improve the performance of this trapping system.     

Changes in the sexual dimorphism of triatominae in the transition from natural to artificial habitats.

A shift from large to small average sizes was observed in Triatoma infestans and Rhodnius domesticus between field and domestic (or laboratory) conditions of life. It was more pronounced in the female specimens, leading to a subsequent reduction of sexual size dimorphism. This feature is discussed in terms of genetic and populational changes occurring from natural to artificial habitats, in particular those related to population densities. Sexual size dimorphism is then recommended as a new character to be used in the study of species of Triatominae adapting to domestic ecotopes.     

Monitoring the domiciliary and peridomiciliary invasion process of Triatoma rubrovaria in the State of Rio Grande do Sul, Brazil

The presence of Triatoma rubrovaria in Brazil has only been confirmed in the States of Paraná and Rio Grande do Sul (RS), where it is found naturally infected with Trypanosoma cruzi. In the wild environment it occurs in rocky habitats and has an eclectic diet, feeding from cockroaches, reptiles and mammals. Data from the Chagas Disease Control Program obtained by the Funda??o Nacional de Saúde, between 1975 and 1997, indicate a growing domiciliary and peridomiciliary invasion of T. rubrovaria in RS, where it has become the most frequently Triatominae species captured in this state since the control of Triatoma infestans. In order to monitor this process, we analyzed collection data derived from 22 years of control campaigns against T. infestans. Collection data for triatomines from domestic habitats show an inverse relationship, with high numbers of T. infestans and low numbers of T. rubrovaria during 1976-1987, compared to the following ten years, 1986-1997, when the number of T. infestans dropped drastically and that of T. rubrovaria increased. There are no consistent indications of intradomiciliary colonization by T. rubrovaria, since only low numbers of nymphs have been captured in the intradomiciliary ecotopes. Nevertheless, this species appears to have preadaptive characteristics for anthropic ecotopes, and should be kept under constant epidemiological surveillance.     

Direct methods for detecting picorna-like virus from dead and alive triatomine insects

In this work we report four different destructive and non-destructive methods for detecting picorna-like virus particles in triatomines. The methods are based on direct observation under transmission electron microscope and they consist of four ways to prepare samples of presumable infected material. The samples are prepared processing dead or alive insect parts, or even dry or fresh insect feces. The methods can be used as analytical or preparative techniques, for quantifying virus infection and checking virus integrity as well. In this work the four methods are applied in order to detect Triatoma virus (TrV) particles in T. infestans colonies.     

Epidemiology of Chagas disease in Ecuador. A brief review

Chagas disease is a complex public health problem that has been underestimated in Ecuador. Here we review the relevant published information, and present unpublished and new data that help to understand the current Chagas disease epidemiological situation and its evolution in the country. Three main characteristics have been identified: (i) persistence of Trypanosoma cruzi transmission in already known foci; (ii) a marked endemicity in some urban areas of Guayaquil; and (iii) the transformation of new Amazon foci into truly endemic areas. The situation in other suspect areas remains uncertain. Five Triatominae species have been implicated in the transmission of T. cruzi to people in Ecuador (Triatoma dimidiata, Rhodnius ecuadoriensis, R. pictipes, R. robustus and Panstrongylus geniculatus), but some others may also play a role in some areas (P. rufotuberculatus, P. howardi, T. carrioni and P. chinai). Other Triatominae reported seem to have little or no epidemiological relevance (T. venosa, T. dispar, Eratyrus mucronatus, E. cuspidatus, P. lignarius and Cavernicola pilosa). High frequency of acute cases and severe chronic disease has been observed. Although cardiomyopathy is more frequent, serious digestive disease is also present. It is estimated that around 120,000-200,000 people may be infected. 2.2 to 3.8 million people are estimated to live under transmission risk conditions.     

The biology of three Mexican-American species of Triatominae (Hemiptera: Reduviidae): Triatoma recurva, Triatoma protracta and Triatoma rubida

The values of biological parameters related to hatching, lifespan, the number of blood meals between moults, mortality, time lapse before the beginning of feeding, feeding time and defecation delay for each instar of three Mexican-American species of Triatominae, Triatoma recurva, Triatoma protracta (former subspecies protracta) and Triatoma rubida (former subspecies uhleri), were evaluated and compared. No significant (p > 0.05) differences were recorded among the three species with respect to the average time required to hatch. This time was approximately 19 days. The average egg-to-adult development time was significantly (p < 0.05) shorter for T. rubida. The number of blood meals at each nymphal instar varied from one-five for each species. The mortality rates were higher for the first-instar nymphs of the three species studied. The mean time lapse before the beginning of feeding was between 0.3-3 min for most nymphs of all instars of each species studied. The mean feeding time was the longest for T. recurva, followed by T. protracta. The defecation delay was less than 10 min for T. recurva and T. rubida. Given these results, only T. rubida should be considered an important potential vector of Trypanosoma cruzi transmission to humans in areas of Mexico where these species exist, whereas T. recurva and T. protracta would be of secondary importance.     

Biology of three species of North American Triatominae (Hemiptera: Reduviidae: Triatominae) fed on rabbits

Aspects related to hatching, lifetime, number of blood meals for molting, mortality, feeding time, and postfeeding defecation delay were evaluated and compared in each instar of three North American Triatominae: Triatoma gerstaeckeri, Triatoma lecticularia and Triatoma protracta, all of them fed on rabbits. No significant differences (p > 0.05) were found among the three species regarding mean hatching rate, which was close to 20 days. Egg-to-adult development times were significantly shorter (p < 0.05) for T. lecticularia. Number of blood meals for molting to next instar ranged from one to five for T. protracta, and from one to six for T. gerstaeckeri and T. lecticularia. Mortality rates were higher in younger nymphs of T. lecticularia and T. protracta, while rates in T. gerstaeckeri were higher in fifth-instar nymphs. Mean feeding time was longest in T. gerstaeckeri, followed by T. lecticularia. More than twice the number of T. gerstaeckeri nymphs completed the development process, if compared to the nymphs from the other two species. Defecation delay was less than 10 min for T. lecticularia, T. protracta and the youngest nymphs of T. gerstaeckeri. Results point out that these three species may be important potential vectors of Trypanosoma cruzi for human populations, in areas of Mexico where these species are currently present.     

The cuticular hydrocarbons of the Triatoma sordida species subcomplex (Hemiptera: Reduviidae)

The cuticular hydrocarbons of the Triatoma sordida subcomplex (Hemiptera: Reduviidae: Triatominae) were ana-lysed by gas chromatography and their structures identified by mass spectrometry. They comprised mostly n-alkanes and methyl-branched alkanes with one-four methyl substitutions. n-alkanes consisted of a homologous series from C21-C33 and represented 33-45% of the hydrocarbon fraction; n-C29 was the major component. Methyl-branched alkanes showed alkyl chains from C24-C43. High molecular weight dimethyl and trimethylalkanes (from C35-C39) represented most of the methyl-branched fraction. A few tetramethylalkanes were also detected, comprising mostly even-numbered chains. Several components such as odd-numbered 3-methylalkanes, dimethylalkanes and trimethylalkanes of C37 and C39 showed patterns of variation that allowed the differentiation of the species and populations studied. Triatoma guasayana and Triatoma patagonica showed the most distinct hydrocarbon patterns within the subcomplex. The T. sordida populations from Brazil and Argentina showed significantly different hydrocarbon profiles that posed concerns regarding the homogeneity of the species. Triatoma garciabesi had a more complex hydrocarbon pattern, but it shared some similarity with T. sordida. The quantitative and qualitative variations in the cuticular hydrocarbons may help to elucidate the relationships between species and populations of this insect group.     

Phylogeny and biogeography of Triatominae (Hemiptera: Reduviidae): molecular evidence of a New World origin of the Asiatic clade

The most representative sample of molecular data, especially 16S and 12S rDNAs, is used to study the phylogeny and evolution of 57 species of three tribes, Rhodniini, Linshcosteini, and Triatomini, of the subfamily Triatominae. For the first time both New World and Old World species are brought together in a single phylogenetic analysis. Maximum-parsimony and distance estimation place both the Asiatic representatives, Linshcosteus and Triatoma rubrofasciata, as sister groups. The Linshcosteus-T. rubrofasciata clade nests firmly within Triatomini, in most analyses branching as a basalmost lineage, thus supporting a monophyletic origin of Triatominae. A paraphyly of "Triatoma" with respect to Linshcosteus, Dipetalogaster, Eratyrus, and Panstrongylus and the paraphyly of "Rhodnius" with respect to Psammolestes is observed in most of the analyses. Reinterpretation of triatomine biogeography points to the origin of Triatominae in northern areas of South America, in Central America, or in the southern region of North America. A few taxonomic changes are proposed: (1) reinclusion of Linshcosteus in Triatomini, (2) inclusion of Psammolestes in Rhodnius, (3) elevation of the "T. flavida complex" to the full genus Nesotriatoma (including N. flavida, N. bruneri, and N. obscura), (4) inclusion of the "T. spinolai complex" in Mepraia (including M. spinolai, M. gajardoi, M. eratyrusiformis, and M. breyeri), and (5) inclusion of "T." dimidiata in Meccus (M. dimidiatus).