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.     

Determinants of the domiciliary density of Triatoma infestans, vector of Chagas disease

In two heavily infested rural villages of Santiago del Estero, Argentina, where no indoor-spraying with residual insecticides had ever been carried out by official control services, we studied the influence of roof and wall structure, domestic use of insecticide, family size and the number of domestic dogs, on the domiciliary density of Triatoma infestans (Klug). Bug density was significantly associated with (1) the interaction between insecticide use and type of roof, (2) the structure of indoor walls, (3) the number of dogs sharing sleeping areas of people (room-mate dogs), and (4) the number of people plus room-mate dogs, but not with just the number of people resident in the house. The interaction between insecticide use and a roof made of 'simbol', a locally available grass (Pennisetum sp.), also reflected a younger age structure of domestic bug populations. In infested houses, the density of bugs infected with Trypanosoma cruzi Chagas was significantly correlated with overall bug density. Our data suggest that the application of environmental management measures by the affected people, such as plastering of walls and modification of roofs, coupled with keeping dogs away from bedrooms and application of insecticides, should limit the domestic population density of T. infestans and thus reduce the transmission of T. cruzi to people.     

Genetic transformation of a Corynebacterial symbiont from the Chagas disease vector Triatoma infestans

Insect-borne diseases have experienced a troubling resurgence in recent years. Emergence of resistance to pesticides greatly hampers control efforts. Paratransgenesis, or the genetic transformation of bacterial symbionts of disease vectors, is an alternative to traditional approaches. Previously, we developed paratransgenic lines of Rhodnius prolixus, a vector of Chagas disease in Central America. Here, we report identification of a Corynebacterial species as a symbiont of Triatoma infestans, a leading vector of Chagas disease in South America. We have modified this bacterium to produce an immunologically active single chain antibody fragment, termed rDB3. This study establishes the basis for generating paratransgenic T. infestans as a strategy for control of Chagas disease.     

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease

Triatoma infestans is a hemiptera, vector of Chagas' disease that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its SG was randomly sequenced. Also, salivary proteins were submitted to two-dimensional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.     

A new method for forensic DNA analysis of the blood meal in chagas disease vectors demonstrated using Triatoma infestans from Chuquisaca, Bolivia

Background

Feeding patterns of the vector are important in the epidemiology of Chagas disease, the leading cause of heart disease in Latin America. Chagas disease is caused by the parasite, Trypanasoma cruzi, which is transmitted by blood feeding insects. Historically, feeding behaviours of haematophagous insects have been investigated using serological reactions, which have detection limits in terms of both taxonomic resolution, and quantity and quality of the blood meal. They are labor intensive, require technical expertise, need fresh or frozen samples and antibodies often are either not available commercially or the resources for synthesis and purification are not available. We describe an assay to identify vertebrate blood meal sources, and the parasite T. cruzi using species-specific PCR assays from insect vectors and use the method to provide information regarding three questions: (1) Do domestic and peri-domestic (chicken coop and animal corral) habitats vary in the blood meals detected in the vectors? (2) What is the pattern of multiple blood meals? (3) Does the rate of T. cruzi infection vary among habitats and is it associated with specific blood meal types?

Methodology/Principal Findings

Assays based on the polymerase chain reaction were evaluated for identification of the blood meal source in the heamatophagous Chagas disease vector Triatoma infestans. We evaluate a technique to identify 11 potential vertebrate food sources from the complex mixture extracted from the vector''s abdomen. We tested the assay on 81 T. infestans specimens collected from the Andean highlands in the department of Chuquisaca, located in central Bolivia, one of the regions in South America where sylvatic T. infestans have been reported. This area is suggested to be the geographic origin of T. infestans and has very high human infection rates that may be related to sylvatic vector populations.

Conclusion/Significance

The results of the assays revealed that a high percentage of insects collected in human dwellings had fed on peri-domestic animals. In contrast, one insect from a chicken coop but no bugs from corrals tested positive for human blood. Forty-eight percent of insects tested positive for more than one vertebrate species. T. cruzi infection was detected in 42% of the specimens. From the epidemiological point of view, the results reveal an overall pattern of movement from peri-domestic structures to human habitations for T. infestans in this region of Bolivia as well as the important role of pigs, dogs, chickens and guinea pigs in the dynamics of T. cruzi infection.     

Population dynamics and feeding behavior of Triatoma brasiliensis and Triatoma pseudomaculata, main vectors of chagas disease in Northeastern Brazil

Biological parameters of Triatoma brasiliensis and T. pseudomaculata that could influence the epidemiological importance of these insects as vectors of Trypanosoma cruzi were compared. The parameters studied were incubation period, interval between hatching or moulting and first feeding, number of blood meals, development time, mortality, net reproductive rate, instantaneous daily reproductive rate, time-lapse before starting feeding, duration of feeding, blood ingestion capacity, occurrence of defecation and blood ingestion velocity. Most aspects of feeding were similar for the two species, although T. pseudomaculata had a longer life cycle than T. brasiliensis producing one and two generations per year, respectively. The two species had similar instantaneous daily rates of population growth.     

Infestin,a thrombin inhibitor presents in Triatoma infestans midgut,a Chagas' disease vector: gene cloning,expression and characterization of the inhibitor

This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively).     

The epidemiologic importance of Triatoma brasiliensis as a chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999

To clarify the epidemiologic importance of Triatoma brasiliensis, the most important Chagas disease vector in the Northeastern of Brazil, capture data related to this species, its distribution, capture index, and percentages of natural infection by Trypanosoma cruzi were examined in 12 different Brazilian states. The Brazilian National Health Foundation collected these data from 1993 to 1999, a period during which a total of 1,591,280 triatomines (21 species) were captured in domiciles within the geographic range of T. brasiliensis. Of this total, 422,965 (26.6%) were T. brasiliensis, 99.8% of which were collected in six states, and 54% in only one state (Ceará). The percentage of bugs infected with T. cruzi varied significantly among states, ranging from 0% (Goiás, Maranh?o, Sergipe, and Tocantins) to more than 3% (Alagoas, Minas Gerais, and Rio Grande do Norte) with an average of 1.3%. This latter value represents a dramatic reduction in the natural infection percentages since 1983 (6.7%) suggesting that, despite the impossibility of eradicating this native species, the control measures have significantly reduced the risk of transmission. However, the wide geographic distribution of T. brasiliensis, its high incidence observed in some states, and its variable percentages of natural infection by T. cruzi indicate the need for sustained entomological surveillance and continuous control measures against this vector.     

Triatoma infestans in Greater Buenos Aires, Argentina

The Health Administration Agencies of many municipalities in Greater Buenos Aires (GBA) receive frequent reports on triatomines in houses. The aim of this work was to identify and describe the dispersal foci of Triatoma infestans in an urban neighborhood of GBA, and contribute to the knowledge of the epidemiological situation in the region. In June 1998, potentially infested places were entomologically evaluated. T. infestans was only detected in a hen building for egg production, which housed approximately 6,000 birds. A total of 2,930 insects were collected. Density was about 9 triatomines/m(2). The proportions of fifth instar nymphs and adults were significantly higher than those of the other stages (p<0.001). The number of triatomines collected largely exceeded the highest domestic infestation found in one house from rural endemic areas of Argentina. Though triatomines were negative for Trypanosoma cruzi, they could acquire the parasite by coming in contact with infected people living in GBA. Besides, the numerous and widely distributed places housing hens and chickens, would favor the settlement of the vector. Together, both facts may constitute a risk of parasitic vectorial transmission. It is recommended to intensify systematic activities of vector search and case detection in GBA.     

The response of the blood-sucking bug Triatoma infestans to carbon dioxide and other host odours

Behavioural responses of Triatoma infestans larvae to carbon dioxide and other odours of vertebrate origin were investigated in a locomotion compensator. T. infestans oriented towards airstreams enriched with carbon dioxide exhibiting a threshold response between 300 and 400 p.p.m. above the ambient CO(2) background. The accuracy of the oriented response to carbon dioxide improved with stimulus intensity. Remarkably, insects did not show any change in their sensitivity threshold to carbon dioxide with the starvation time. The attractiveness to carbon dioxide depended on the time of the day, i.e. these nocturnal bugs only oriented towards carbon dioxide-loaded airstreams during the first hours of the scotophase. L-lactic acid did not evoke oriented responses when it was presented as a single stimulus in a wide range of intensities. However, a marked synergism was evident when L-lactic acid was combined with a sub-threshold concentration of carbon dioxide. Under this condition, the threshold response to carbon dioxide decreased to 75-150 p.p.m. above ambient CO(2) background. The isomer D-lactic acid evoked no response, either alone or in combination with carbon dioxide. When insects were stimulated with 1-octen-3-ol a significant positive orientation was found. This response was not modified by the addition of carbon dioxide.     

Scanning electron microscopic studies of Trypanosoma cruzi in the rectum of its vector Triatoma infestans

The cuticular surface of the rectum of Triatoma infestans and its colonization by a Trypanosoma cruzi strain originating from the same locality as the bugs were studied by scanning electron microscopy at different weeks post infectionem. On the basis of the cuticular folding, the rectum can be subdivided into five regions. The rectal gland has the finest structure and the region anterior to the anus the deepest folds. Try. cruzi always prefers to colonize the rectal gland, while the other regions are colonized in varying densities. Most of the flagellates are epimastigotes (long and short), except in the region at the entrance of the midgut, where trypomastigotes predominate.     

Trypanosoma cruzi: origin of metacyclic trypomastigotes in the urine of the vector Triatoma infestans

Population density and percentage of the different stages of an established infection of Trypanosoma cruzi were determined for two parts of the excretory system and for the rectum of fifth instars of Triatoma infestans unfed and 4 hr after feeding. These data were also evaluated for feces and urine of the fed bugs. In the first unfed group only small populations of the flagellate occurred in the Malpighian tubules and ampullae and not in all bugs. The three rectal populations (rectal lumen and anterior and posterior rectal wall) consisted of approximately equal numbers. About 10% were spheromastigotes and about 10% were stages intermediate to epimastigotes. Significantly fewer epimastigotes and more trypomastigotes were present on the rectal wall than in the lumen. Two intermediate forms leading to the trypomastigote stage occurred in similar numbers. In nearly all bugs the initial excretion (feces) contained the highest number of flagellates as compared to the following drops of urine. More flagellates were excreted through the urine than were contained in the excretory system of unfed bugs. The population in the feces reflected the percentage of forms present in the rectal lumen of unfed bugs, but in the urine the percentage of trypomastigotes increased up to 100%. Four hours after blood uptake, dissection of bugs still showed parasites in the Malpighian tubules and ampullae; the total number of parasites in the rectum was reduced by more than 50%. This reduction was more pronounced in the rectal lumen and on the posterior rectal wall. In stained smears from all three rectal populations there were rarely spheromastigotes but high percentages of epimastigotes. The intermediate stages leading to trypomastigotes mainly originated from short epimastigotes. Comparison of the T. cruzi populations before and after feeding demonstrates that the trypomastigotes in the urine should originate from the rectal wall, especially from the posterior part.     

Microsatellites reveal a high population structure in Triatoma infestans from Chuquisaca, Bolivia

Background

For Chagas disease, the most serious infectious disease in the Americas, effective disease control depends on elimination of vectors through spraying with insecticides. Molecular genetic research can help vector control programs by identifying and characterizing vector populations and then developing effective intervention strategies.

Methods and Findings

The population genetic structure of Triatoma infestans (Hemiptera: Reduviidae), the main vector of Chagas disease in Bolivia, was investigated using a hierarchical sampling strategy. A total of 230 adults and nymphs from 23 localities throughout the department of Chuquisaca in Southern Bolivia were analyzed at ten microsatellite loci. Population structure, estimated using analysis of molecular variance (AMOVA) to estimate F_ST (infinite alleles model) and R_ST (stepwise mutation model), was significant between western and eastern regions within Chuquisaca and between insects collected in domestic and peri-domestic habitats. Genetic differentiation at three different hierarchical geographic levels was significant, even in the case of adjacent households within a single locality (R _ST = 0.14, F _ST = 0.07). On the largest geographic scale, among five communities up to 100 km apart, R _ST = 0.12 and F _ST = 0.06. Cluster analysis combined with assignment tests identified five clusters within the five communities.

Conclusions

Some houses are colonized by insects from several genetic clusters after spraying, whereas other households are colonized predominately by insects from a single cluster. Significant population structure, measured by both R _ST and F _ST, supports the hypothesis of poor dispersal ability and/or reduced migration of T. infestans. The high degree of genetic structure at small geographic scales, inferences from cluster analysis and assignment tests, and demographic data suggest reinfesting vectors are coming from nearby and from recrudescence (hatching of eggs that were laid before insecticide spraying). Suggestions for using these results in vector control strategies are made.     

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS–PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K_i = 3.29 nM) and human cathepsin L (K_i = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.     

Triapsin, an unusual activatable serine protease from the saliva of the hematophagous vector of Chagas' disease Triatoma infestans (Hemiptera: Reduviidae)

Salivary anticoagulant activities are widely distributed among hematophagous arthropods. Most of them are inhibitors of the serine proteases of the coagulation cascade. Here we show that the saliva of the exclusively hematophagous insect Triatoma infestans, an important vector in the transmission of Chagas' disease, contains an uncommon trypsin-like activity, triapsin. This novel enzyme was purified and characterized. It is a serine protease that is stored as a zymogen in the luminal content of the salivary glands D2. Triapsin is activated by trypsin treatment, or when the saliva is ejected during the insect bite. The enzyme was purified 300-fold from the released saliva by anion exchange chromatography in a HiTrap Q column, followed by chromatography in Phenyl-Superose, and Superdex HR75. The purified triapsin shows an apparent molecular mass of around 40 kDa in non-reduced SDS gels and in sieving chromatography, and 33 kDa in reduced SDS-gels. Its activity is lost after incubation with dithiothreitol indicating that cysteine bridges are essential for activity. Triapsin cleaves gelatin and synthetic substrates showing preference for arginine at P1 residues. The best p-nitroanilide substrate is isoleucyl-prolyl-arginine. It does not cleave bradykinin, angiotensin and other lysine containing substrates. The triapsin amidolytic activity against chromogenic substrates is similar to plasminogen activators, such as urokinase and tissue plasminogen activator. However, it does not activate plasminogen. The fact that triapsin is released at the bite in its active form suggests that it has a role in blood feeding.     

Comparison of feeding behaviour of Triatoma infestans, Triatoma brasiliensis and Triatoma pseudomaculata in different hosts by electronic monitoring of the cibarial pump

Feeding behaviour of Triatoma infestans, T. brasiliensis and T. pseudomaculata on pigeons and mice was compared by electronic monitoring of the cibarial pump. The methodology developed permits the study detailed of triatomine feeding behaviour using an artificial feeder as well as on live hosts. T. infestans was the species that fed most rapidly on the two hosts tested (28.03+/-1.6 mg/min for pigeon and 21.33+/-1.7 mg/min for mouse), followed by T. brasiliensis (17.09+/-1.4 mg/min and 13.1+/-1.5 mg/min for pigeon and mouse, respectively) and T. pseudomaculata, (5.23+/-0.6 mg/min and 4.09+/-0.4 mg/min for pigeon and mouse, respectively). The quantity of liquid ingested per stroke of the cibarial pump was 100.7+/-4.1 nl for T. infestans, 69.1+/-2.7 nl for T. brasiliensis and 26.8+/-1.5 nl for T. pseudomaculata. The rate of engorgement in pigeons tended to be greater than that obtained for mice in the three species studied. In the experiments carried out using mice, probing times tended to be longer and interruptions during the meal more prolonged. This reinforces the idea that feeding on mice is more difficult than on pigeons, requiring more contact time to obtain the similar quantity of blood.     

Characterization of Guinea Pig Antibody Responses to Salivary Proteins of Triatoma infestans for the Development of a Triatomine Exposure Marker

Background

Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure.

Methodology and Principal Findings

In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5^th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5^th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs.

Conclusion

Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential antigen as useful marker of T. infestans exposure.     

Strategies of soilborne plant pathogenic fungi in relation to disease suppression

The purpose of classifying pathogens based on ecological traits is that certain generalizations can be made beyond the specific or “model” pathogen that has been investigated. Usually this is done at the taxonomic level of species, but infraspecific variation in ecological traits frustrates such classifications. Moreover, shifts in traits can take place during the life cycle of the pathogen, and plant-mediated rhizosphere effects affect the ecological traits of pathogens. It is therefore useful to separate management of the pathogen, leading to pathogen suppression, from management of the disease, which is essentially plant-mediated. Both approaches may act via specific or general suppression, although the latter mainly acts in the bulk soil. An ecological classification of a given pathogen isolate needs to take into account host range, host sensitivity, host species (in relation to systemically acquired resistance (SAR)/induced systemic resistance (ISR) and host-affected rhizosphere communities), sensitivity to fungistasis, ability to compete for organic matter, sensitivity to specific disease suppression and survival capability. We conclude that generalization at the species level is imprecise and often misleading.     

Circadian rhythm of behavioural responsiveness to carbon dioxide in the blood-sucking bug Triatoma infestans (Heteroptera: Reduviidae)

The temporal modulation of the behavioural response to carbon dioxide and its chronobiological basis were investigated in larvae of Triatoma infestans. We analysed the orientation towards CO(2) of insects kept under three different illumination regimes: (1) 12 h light/12 h darkness cycles (L/D), (2) constant darkness (D/D) and (3) constant light (L/L). When maintained under L/D conditions, insects exhibited an oriented response towards airstreams added with 1500 ppm of CO(2) during the first hours of the scotophase only. Bugs maintained under D/D also showed a positive orientation response towards CO(2) during the first hours of the subjective night, while bugs kept under L/L did not show a rhythmic oriented behaviour. Thus, T. infestans displayed a daily rhythm of orientation towards CO(2) (i.e. a potential food source) only at the beginning of the scotophase. The persistence of the rhythm under constant darkness reveals the existence of an endogenous circadian control of this behaviour.