Chagas disease remains a public health concern in Brazil and other Latin American countries, mainly due to the potential domiciliation of native triatomine species. We analyzed the genetic variability of Triatoma pseudomaculata in sylvatic and peridomestic ecotopes throughout three localities in the northeastern state of Bahia, Brazil. We studied polymorphisms generated by random amplified polymorphic DNA (RAPD) and isoenzyme electrophoresis analyses. Based on RAPD analysis, each specimen was assigned to one of three genetic clusters. Although all sylvatic specimens from one locality were grouped into the same cluster, sylvatic and peridomestic specimens from the other two localities were broadly distributed between the remaining two clusters, suggesting that geographic population structuring was not occurring. Furthermore, isoenzyme analysis suggested that distinct populations were in Hardy-Weinberg equilibrium. Low statistical values for Wright's Fst index also supported the absence of population structuring and suggested the occurrence of panmixia. We conclude that genetic flow occurs between sylvatic and peridomestic T. pseudomaculata populations, probably as a consequence of passive and active dispersion of the insects, associated with deforestation and anthropic transformations.
Chagas disease (American trypanosomiasis), caused by infection with the flagellate protozoan Trypanosoma cruzi and transmitted by reduviid bugs, remains a public health concern in Brazil and other Latin American countries (Dias 2007). Despite successful chemical control of Triatoma infestans through an initiative carried out by the Southern Cone countries, indoor and peridomestic invasion and colonization of wild triatomine species may favor a new epidemiological scenario for Chagas disease (Costa and Lorenzo 2009, Guhl et al. 2009, Fé et al. 2009). In this context, the originally arboreal species Triatoma pseudomaculata may assume particular importance. T. pseudomaculata has been found infesting peridomestic structures and invading dwellings in northeastern Brazil. In the peridomestic environment, T. pseudomaculata preferred to colonize wood perches and woodpiles (Sarquis et al. 2004, Sarquis et al. 2006). Also, it was demonstrated that even after eight years of entomological surveillance in an area in the Jequitinhonha Valley, bordering the state of Bahia, T. pseudomaculata specimens were found in domiciliary units (de Assis et al. 2007). In nature, T. pseudomaculata is found in bird nests and many species of trees. Although apparent preference for any particular tree species has not been recorded (Carbajal de la Fuente et al. 2008), an association with the black acacia bush (Mimosa tenuiflora) was noted in areas suffering anthropic transformation and deforestation (Freitas et al. 2004).
Since the 1960s, the initial entomological surveys carried out in northeastern Brazil demonstrated the epidemiological importance of T. pseudomaculata. In the state of Ceará, T. pseudomaculata was captured in 68.8% of the municipalities (Alencar and Sherlock 1962). These data support the evidence that T. pseudomaculata can be well adapted to artificial and anthropic ecotopes; furthermore, this species can present a high natural T. cruzi infection rate (15 to 18%) (Sarquis et al. 2004, Carbajal de la Fuente et al. 2008). Triatoma pseudomaculata is distributed throughout central and northeastern Brazil, especially the semi-arid (caatinga) zones (Carcavallo et al. 1999).
Unfortunately, while some crucial steps in the control of autochthonous species are not routinely performed, like the improvement of the domestic and peridomestic environments which could prevent re-infestation by T. pseudomaculata and other native species, control measures must be based in the periodical chemical treatment of dwellings. In this context, the assessment of gene flow between sylvatic and peridomestic T. pseudomaculata populations could provide clues for the study of the dispersive capacity of that species. Such data would support vigilance and control actions, including inferences about the founding of domestic colonies and sources of re-infestation during the surveillance phase of control programs, as proposed for T. infestans and T. sordida in Bolivia, and the population structuring within specific geographic settings (Noireau et al. 1999b, Dujardin et al. 1998, Dujardin et al. 1997, Pacheco et al. 2003, Pacheco et al. 2007, Borges et al. 2005).
This work attempted to assess, on the genetic level, the dispersion of T. pseudomaculata throughout sylvatic and peridomiciliary ecotopes within an area of northeastern Brazil. Population geographic structuring was evaluated by comparing observed and expected frequencies of multilocus isoenzyme genotypes and polymorphisms generated by random amplified polymorphic DNA (RAPD).
MATERIALS AND METHODS
Study areas and triatomine capture
Curaçá municipality (8º 58’ S / 39º 53’ W) is situated on the banks of the São Francisco River, in the state of Bahia, northeastern Brazil (Figure 1). It belongs to the semi-arid Caatinga biome, which comprises about 10% of Brazil. The local population practices subsistence agriculture and rudimentary cattle-raising. Triatomines were sought in peridomestic structures, with prior consent of the dwellers, and in sylvatic ecotopes (hollow trees) using live-bait traps (Noireau et al. 1999a). Three areas were investigated, each area encompassing ∼ 1 km2: i) Serra do Icó (SI), a sylvatic area situated 8 km from Curaçá; ii) Baixa do Angico (BA), located 21 km from Curaçá; and iii) Santo Antonio (SA), 50 km from Curaçá. Triatomines were captured from the following ecotopes in each area: SI, sylvatic; BA, sylvatic and peridomestic (mainly chicken huts); and SA, peridomestic (chicken huts and corrals). Each analyzed specimen came from distinct sites of collection (trees or peridomestic structures).
RAPD and numerical (phenetic) analyses
Twenty-eight specimens were investigated by RAPD analysis (Table 1). DNA was extracted from the legs of each specimen (DNA Genomic Prep, Amersham Pharmacia Biotech) followed by additional phenol-chloroform purification. DNA concentrations and purity were determined by agarose gel electrophoresis and comparison with DNA standards of known concentration. Amplification was carried out with five decameric primers from the Ready-To-Go RAPD analysis kit (Amersham Pharmacia Biotech, Little Chalfont, U.K.). For PCR amplification, cycling conditions previously described (Pacheco et al. 2003) were performed using a programmable thermal cycler (GeneAmp PCR System 9600, Applied Biosystems, Foster City, CA). Amplification products were subjected to electrophoresis in 2% agarose gels in 1 × Tris-borate-EDTA buffer, stained with ethidium bromide and recorded on a gel documentation system (UVP, Upland, CA) with ultraviolet trans-illumination. An out-group (DNA from Triatoma sordida) and a negative control were included in each gel. In addition, one sylvatic specimen of T. pseudomaculata from Sobral, state of Ceará, was also included in the analysis. To determine the proportion of mismatched bands between pairs of T. pseudomaculata, phenetic analysis was performed using the Simple Matching Coefficient of Similarity. The similarity matrix was transformed into a dendrogram using the UPGMA algorithm (Sneath and Sokal 1973) with NTSys version 2.0 software (Exeter Software, Setauket, NY).
Table 1. Locality of origin of Triatoma pseudomaculata specimens in the municipality of Curaçá, Bahia.
RAPD Random Amplified Polymorphic DNA.
MLEE – Multilocus Enzyme Electrophoresis.
Serra do Icó
Baixo do Angico
Isoenzyme electrophoresis analyses
Seventy-two insects were analyzed (Table 1). Thoracic muscles were dissected and ground in 100 ml of an enzyme stabilizer (dithiothreitol, E-aminocaproic acid, and EDTA, each at 2 mM). Extracts were stored at −70° C prior to use. Isoenzyme analysis was performed on cellulose acetate plates (Helena Laboratories, Beaumont, TX). The following 16 enzyme systems were assayed: aconitate hydratase (ACON, EC 18.104.22.168); diaphorase (DIA, EC 22.214.171.124); fructose-1, 6-diphosphatase (FDP, EC 126.96.36.199); fumarate hydratase (FUM, EC 188.8.131.52); glutamate dehydrogenase (GDH, EC 184.108.40.206); aspartate aminotransferase (GOT, EC 220.127.116.11); glycerol-3-phosphate dehydrogenase (GPD, EC 18.104.22.168); glucose phosphate isomerase (GPI, EC 22.214.171.124); glucose-6-phosphate dehydrogenase (G6PD, EC 126.96.36.199); hexokinase (HK, EC 188.8.131.52); isocitrate dehydrogenase (IDH, EC 184.108.40.206); malate dehydrogenase (MDH, EC 220.127.116.11); malic enzyme (ME, EC 18.104.22.168); mannose-phosphate isomerase (MPI, EC 22.214.171.124); phosphoglucomutase (PGM, EC 126.96.36.199); and 6-phosphogluconate dehydrogenase (6-PGDH, EC 188.8.131.52). Electrophoresis and enzyme staining were performed as described previously by Noireau et al. (1998). Genotype distribution was obtained by direct interpretation of gel banding patterns. Analysis of spatial subdivision of T. pseudomaculata sub-populations was performed for polymorphic loci. The index F (Nei 1978), which measures the departure from panmixia of individuals relative to the sub-populations, and the index Fst (Wright 1978), which measures the gene frequencies differences among sub-populations, were calculated.
Genetic heterogeneity was detected by RAPD analysis of the triatomines. Of five decameric primers tested, three (Primer 1, 5’-GGTGCGCGGA-3’; Primer 2, 5’-GTTTCGCTCC-3’; and Primer 4, 5’-AAGAGCCCGT-3’) revealed different degrees of intraspecific genetic polymorphism by generating distinct banding patterns for the studied specimens. Profiles generated by these primers produced a total of 33 characters, with amplification products ranging from 300 to 1,200 base pairs (bp). Based on phenetic analysis, each of the 28 specimens was placed into one of three major clusters (Figure 2). The first cluster was comprised of all sylvatic specimens from Serra do Icó and the specimen collected in Sobral, State of Ceará. The second phenetic cluster contained all peridomestic and two sylvatic specimens from Baixo do Angico, in addition to two peridomestic triatomines from Santo Antonio. In this group, two specimens from Baixo do Angico (154BA/P and 158BA/P) were genetically similar, sharing 91% of common characteristics. Finally, the third cluster contained three sylvatic and six peridomestic specimens collected in Baixo do Angico and Santo Antonio, respectively (Figure 2). Bootstrap values were not determined in this study, however, phenetic analyses using a different coefficient of similarity (Jaccard's Coefficient) were performed corroborating the results (data not shown).
From a total of 16 enzymatic systems, only two loci (Acon and 6pgdh) were polymorphic (Table 2). The locus Acon could not be assessed in five of the sylvatic insects from Serra do Icó probably due to loss of enzyme activity during procedures. The genotype frequencies for the polymorphic loci among the studied sub-populations showed no significant departures from the expectations of the Hardy-Weinberg equilibrium (calculated by the F index of Nei; P > 0.05, (Table 3)). Results obtained with Fst statistics did not show spatial structuring (Table 4). Results provided by isoenzyme analysis were therefore in accordance with RAPD data.
Table 2. Genotype distribution of sub-populations studied in distinct localities in the municipality of Curaçá Bahia, Brazil.
Table 4. Fst index (Wright 1978) at two polymorphic loci (Acon and 6Pgdh) among Triatoma pseudomaculata sub-populations from the three surveyed areas.
* Baixo do Angico (PD + S), San Antonio (PD), and Serra do Icó (S).
** Baixo do Angico (PD), Baixo do Angico (S), San Antonio (PD), and Serra do Icó (S).
One of the most significant challenges to Chagas disease control is that several species of vectors previously considered exclusively sylvatic are now changing their behavior, and domiciliary colonies of those vectors have been recorded (Guhl et al. 2009). In the northeast region of Brazil, this scenario is particularly applicable to two triatomine species, T. brasiliensis and T. pseudomaculata, both native to the Caatinga biome (Silveira et al. 2001).
The RAPD analyses presented here suggest genetic homogeneity between peridomestic and sylvatic T. pseudomaculata specimens and point to the absence of population structuring. However, sylvatic specimens collected in Serra do Icó were restricted to a single phenetic cluster; in this locality, only sylvatic specimens could be collected, so the structuring of sylvatic and peridomestic triatomines could not be assessed. Interestingly, the species from Sobral was grouped in a subgroup within this cluster, with two specimens from Serra do Icó, sharing a coefficient of similarity of 0.74. The geographic isolation of Serra do Icó could explain the clustering of wild specimens collected in this locality. The relatively low genetic variability among the collected specimens allowed us to analyze only two enzymatic loci by multilocus enzyme electrophoresis. Results suggest that all triatomine sub-populations are in Hardy-Weinberg equilibrium. This result corroborates the RAPD data and implies panmixia, supporting the hypothesis that genetic flow occurs among the domestic and sylvatic T. pseudomaculata populations.
The lack of population structuring between sylvatic and peridomestic T. pseudomaculata in the studied area is probably a consequence of passive and/or active dispersal of triatomines from their natural ecotopes towards the peridomestic environment. This suggests that peridomestic populations could be continuously replaced by wild specimens within a dynamic and continuous process of invasion and colonization.
In a scenario of deforestation and anthropic transformations, passive dispersion of sylvatic T. pseudomaculata populations towards the peridomestic environment has been well demonstrated. Freitas et al. (2004) reported the presence of T. pseudomaculata in the phloem of the bush Mimosa tenuiflora and the utilization of its wood for timber and in the construction of corrals. Dwellers also keep this wood accumulated in the peridomicile. Active dispersal of T. pseudomaculata has also been noticed, and the attraction of wild bugs by light was documented. Also, it was observed that triatomines attracted by light appear to have a food deficit (Carbajal de la Fuente et al. 2007). In both cases, the destruction of natural ecotopes and reduction of wild animal blood sources are determinants of the invasion of human peridomiciles by triatomines as they search for blood meals in domestic animals (Sarquis et al. 2006). However, despite the trend of T. pseudomaculata to colonize peridomestic structures like chicken huts and small ruminant corrals, this species is rarely captured inside houses in northeastern Brazil. In an entomological survey, T. pseudomaculata corresponded to 2.5% of all intradomicile captures (Sarquis et al. 2004).
The chemical treatment of peridomiciles that are characterized by a continuous reintroduction of sylvatic specimens of triatomines represents a real challenge to health authorities. In addition, peridomestic structures, like corrals and chicken huts often built with already infested T. pseudomaculata wood, are temporary, being constantly destroyed and reconstructed (Oliveira Filho et al. 2000). This illustrates the difficulties of insecticide-based control measures, which may have low efficacy against this and other species. We argue that actions for chemical control of Chagas disease vectors in such eco-epidemiological scenarios should be strongly focused in the intradomicile, preventing the colonization of the houses by triatomines, including continuous entomological surveillance of the peridomestic environment and improving the physical characteristics of the dwellings.