Effect of salivary CYP4EM1 and CYP4EM2 gene silencing on the life span of Chagas disease vector Rhodnius prolixus (Hemiptera, Reduviidae) exposed to sublethal dose of deltamethrin.

Control of Chagas disease in endemic countries is primarily accomplished through insecticide spraying for triatomine vectors. In this context, pyrethroids are the first-choice insecticide, and the evolution of insect resistance to these insecticides may represent an important barrier to triatomine control. In insects, cytochrome P450s are enzymes involved in the metabolism of xenobiotics and endogenous chemicals that are encoded by genes divided into different families. In this work, we evaluated the role of three Rhodnius prolixus CYP4EM subfamily genes during blood meal and after deltamethrin exposure. CYP4 gene members were expressed in different insect organs (integument, salivary glands (SGs), midgut, fat body and malpighian tubules) at distinct transcriptional levels. CYP4EM1 gene was highly expressed in the SG and was clearly modulated after insect blood meal. Injection of CYP4EM1dsRNA promoted significant reduction in mRNA levels of both CYP4EM1 and CYP4EM2 genes and induced deleterious effects in R. prolixus nymphs subsequently exposed to sublethal doses of deltamethrin (3.4 or 3.8 ng/nymph treated). The higher dose reduced the survival over time and increased susceptibility of R. prolixus nymphs to deltamethrin. A better understanding of this mechanism can help in developing of more efficient strategies to reduce Trypanosoma cruzi vector transmission in Americas.

Misidentification of two Brazilian triatomes, Triatoma arthurneivai and Triatoma wygodzinskyi, revealed by geometric morphometrics.

Triatoma arthurneivai Lent & Martins and Triatoma wygodzinskyi Lent (Hemiptera: Reduviidae) are two Brazilian species found in the sylvatic environment. Several authors may have misidentified T. arthurneivai and consequently published erroneous information. This work reports the use of geometric morphometric analysis on wings in order to differentiate T. arthurneivai and T. wygodzinskyi, and thus to detect possible misidentifications. Triatomines collected from the field in the states of Minas Gerais and São Paulo, and from laboratory colonies, were used. Analyses show a clear differentiation between specimens of T. arthurneivai and T. wygodzinskyi. This indicates that T. arthurneivai populations from São Paulo state were misidentified and should be considered as T. wygodzinskyi. This study also suggests that T. arthurneivai is an endemic species from Serra do Cipó, Minas Gerais state.

The association between the geographic distribution of Triatoma pseudomaculata and Triatoma wygodzinskyi (Hemiptera: Reduviidae) with environmental variables recorded by remote sensors.

In this study, predictive models of geographic distribution patterns of Triatoma pseudomaculata (Tps) and T. wygodzinskyi (Twy) were carried out. They were based on biophysical variables estimated from information provided by the satellite remote sensors AVHRR (Advanced Very High Resolution Radiometer) and MODIS (MODerate-resolution Imaging Spectroradiometer). Our goal was to analyze the potential geographic distribution of Tps and Twy and to assess the performance of three predictive models (one for each species and one for both species together) based on temperature, vapour pressure deficit, vegetation and altitude. The geographic distribution analysis shows that all models performed well (>85.7% of overall correct classification of presence and absence point data). The MODIS-based models showed lower correct classifications than the AVHRR-based models. The results strongly suggest that environmental information provided by remote sensors can be successfully used in studies on the geographic distribution of poorly understood Chagas disease vector species.

Microsatellite markers in Triatoma pseudomaculata (Hemiptera, Reduviidae, Triatominae), Chagas' disease vector in Brazil.

Six polymorphic microsatellite loci were isolated and characterized using a microsatellite-enriched genomic library from the Chagas' disease vector Triatoma pseudomaculata. This species is found in Brasil in Caatinga areas and predominantly in peridomestic habitats. All the microsatellites tested on a population of T. pseudomaculata sampled in the Bahia State, Brazil, were polymorphic (2-15 alleles). Markers amplification was also tested on six Triatoma species and some loci successfully amplified in the most phylogenetically related species, in particular Triatoma brasiliensis.

The use of aggregation signals by Triatoma brasiliensis (Heteroptera: Reduviidae).

Triatoma brasiliensis is considered the main vector of Chagas disease in the semiarid zones of North-eastern Brazil. In this work, we describe the spatial pattern of defecation in T. brasiliensis and the spatial relationship of faeces with the shelters of these bugs. Afterwards, we analyse the attraction response of T. brasiliensis larvae to potential odour cues present in their dry faeces, to substances present in their cuticule (footprints) and to the dry faeces of Triatoma pseudomaculata. We show that faecal spots are deposited in a larger density inside the shelter than in the remaining area available for the bugs. T. brasiliensis larvae were significantly attracted towards their own faeces, and also to those of T. pseudomaculata. In contrast to other triatomine species, footprints did not promote attraction in T. brasiliensis. The potential use of communication signals of T. brasiliensis as tools for helping control this species is discussed.

Identification of morphologically similar Rhodnius species (Hemiptera: Reduviidae: Triatominae) by electrophoresis of salivary heme proteins.

We examined intraspecific variability in the genus Rhodnius using starch gel electrophoresis of salivary heme proteins. Salivary protein profiles of 8 Rhodnius species (R. prolixus, R. robustus, R. neglectus, R. nasutus, R. ecuadoriensis, R. pallescens, R. pictipes, and R. domesticus) were compared. All species could be distinguished by this technique. The greatest protein polymorphism was found in R. ecuadoriensis, R. nasutus, R. robustus, and R. pictipes, followed by R. prolixus, R. neglectus, R. pallescens, and R. domesticus. This approach was able to distinguish R. prolixus from R. robustus and R. neglectus from R. nasutus, species with extreme phenotypical similarity.

Genetic variability and geographic differentiation among three species of Triatomine bugs (Hemiptera-Reduviidae).

Three species of triatomine bugs, Triatoma sordida, T. guasayana, and T. patagonica, were examined by cytogenetic (C-banded karyotypes and male meiotic process) and isoenzymatic studies. These three species, with different importance as Chagas' disease vectors, were found to be closely related according to their known ethologic, ecologic, and morphologic traits. Although they have the same diploid chromosome number (2n = 22 constituted by 20 autosomes and an XY male/XX female sex mechanism), each species has a distinct chromosomal behavior during male meiosis and a specific amount and localization of C-heterochromatic blocks. Moreover, these chromosome characteristics allowed us to differentiate two T. sordida populations. Isoenzymatic data confirmed the taxonomic status of the three species and together with our cytogenetic results questioned the species homogeneity of T. sordida.

Variability of the salivary proteins of 20 Brazilian populations of Panstrongylus megistus (Hemiptera: Reduviidae: Triatominae).

The objective of the present study was to study variability in the salivary proteins of 20 Panstrongylus megistus populations from different ecotopes and verify whether this variability influenced the intensity of the response to specific anti-saliva antibodies. Electrophoretic analysis of P. megistus saliva showed a complex protein composition and great interpopulation variability. A higher concentration of bands was observed in the 17-29 kDa region. The phenogram constructed from the electrophoretic profiles of the P. megistus study populations revealed the existence of two main groups. However, there was no evident relationship between these groups and geographical regions, ecotopes or hosts. Saliva inoculated by P. megistus during feeding elicited production of low level of anti-saliva antibodies in rabbit. The homologous and heterologous salivary proteins were recognised by serum of rabbit sensitised with saliva from only one population. Qualitative and quantitative differences were observed for recognised bands in the saliva of all eight populations studied by Western blot analysis. The most recognised bands were those of greatest molecular weight (68.0-97.4 kDa).

Differences in saliva composition among three Brazilian populations of Panstrongylus megistus (Hemiptera, Reduviidae).

Comparisons of electrophoretic profiles of triatomines (Panstrongylus megistus) from the Brazilian states of Bahia (BA), Minas Gerais (MG) and Santa Catarina (SC) revealed differences in the composition of the saliva between the three populations. A phenogram constructed on the basis of the most representative electrophoretic banding patterns allowed the specimens tested to be divided into two groups. The BA and SC populations could be completely separated while some individuals from MG could be placed in each group. The BA population presented fewer bands than the MG and SC ones. The populations studied also differed with regard to their degree of association with human habitations (sylvatic, peridomiciliary and/or intradomiciliary) and our results suggest that the proteinaceous composition of the saliva of the three distinct populations of triatomines could be related to the hosts each is likely to encounter.

Aggregation behaviour in Panstrongylus megistus and Triatoma infestans: inter and intraspecific responses.

We tested the aggregation response to inter and intraspecific chemical signals in Panstrongylus megistus and Triatoma infestans. As previously described for T. infestans, larvae of P. megistus significantly aggregated on papers impregnated with their own excrement and on papers marked with cuticular substances deposited on surfaces on which these insects had walked. T. infestans bugs also aggregated on papers impregnated by faeces or by cuticular substances of P. megistus, and P. megistus aggregated on papers contaminated by faeces or by cuticular substances of T. infestans. The response of P. megistus to its cuticular substances was significantly stronger than that to its faeces. The non-specificity of the two signals is discussed in the context of the ecological relationship between both species.

Salivary heme proteins distinguish Rhodnius prolixus from Rhodnius robustus (Hemiptera: Reduviidae: Triatominae).

Rhodnius prolixus interpopulation variability was studied based on a new approach using salivary heme proteins (nitrophorins) electrophoresis in starch gel. We compared salivary proteins profiles of R. prolixus from three different laboratory colonies from Honduras, Venezuela, Brazil and Rhodnius robustus from Venezuela, constructing a UPGMA. The Honduran and Venezuelan populations could not be distinguished from each other, but the Brazilian population was well separated from the others. The high similarity between Honduran and Venezuelan specimens lends support to current theories that the Central American populations of R. prolixus may have been introduced from a Venezuelan origin. The low polymorphism shown by the Honduran specimens is in agreement with a possible founder effect. This new approach also distinguished R. prolixus populations from R. robustus, species with extreme phenotypical similarity.

Anticoagulant activity of Triatoma infestans and Panstrongylus megistus saliva (Hemiptera/Triatominae).

Salivary gland extract of Triatoma infestans prolonged thrombin time, prothrombin time, and activated partial thromboplastin time, while salivary gland extract of Panstrongylus megistus delayed thrombin time and activated partial thromboplastin time. However, saliva of P. megistus prolonged activated partial thromboplastin time and saliva of T. infestans delayed activated partial thromboplastin time and prothrombin time. T. infestans saliva interferes particularly with activity of factor VIII (intrinsic pathway) and factor V (common pathway), but also affects other factors. Saliva of the triatomine species studied presented distinct SDS-PAGE profiles. These results demonstrate that there are differences in anticoagulant activity and protein composition of triatomine saliva.

Yeast culture volatiles as attractants for Rhodnius prolixus: electroantennogram responses and captures in yeast-baited traps.

Responses to air currents carrying volatiles from yeast cultures were measured by means of electroantennograms (EAGs) in the haematophagous bug Rhodnius prolixus and we tested yeast-baited traps in the laboratory. The volatiles liberated by yeast cultures generated a clear electrophysiological response, much higher than that obtained during stimulation with clean air. The addition of yeast cultures to the traps dramatically increased overnight capture. A modification performed on the traps was effective for this species, as the bugs were immobilised once they fell into the device. Our results demonstrate that yeast culture volatiles are attractive for R. prolixus. The results obtained by means of electroantennogram techniques represent the first electrophysiological evidence of the perception of yeast volatiles by the antennae of a triatomine bug. We discuss the relevance of our results in view of currently used sampling techniques and control strategies.

Influence of the blood meal source on the development of Triatoma infestans, Triatoma brasiliensis, Triatoma sordida, and Triatoma pseudomaculata (Heteroptera, Reduviidae).

The objective of this study was to determine the influence of the blood meal source on the life cycle and reproductive development of female Triatoma infestans (Klug, 1834), Triatoma brasiliensis Neiva, 1911, Triatoma sordida (Stal, 1859), and Triatoma pseudomaculata Corrga & Espfinola, 1964. In all triatomine species studied the life cycle was shorter for the groups fed on mice than for those fed on pigeons, the range of differences being between 1.5 times (T pseudomaculata and T. infestans) and 2.4 times (T brasiliensis). The mortality rate of nymphs during the life cycle tended to be greater in insects fed on pigeons than in those fed on mice, the differences for T. brasiliensis being statistically significant. Females of T sordida and T pseudomaculata had a greater fecundity than those of T. infestans and T. brasiliensis independently of the blood meal source. The differences of fecundity observed probably reflect differences in the availability of blood in the silvatic ecotopes of these species, meals being more frequent for T. infestans and T brasiliensis, which live at high densities in association with rodents in highly stable ecotopes. Because T. sordida and T. pseudomaculata live in more unstable ecotopes with fewer sources of blood they form small sparse colonies and invest more energy in reproduction than maintenance.

Genetic variability of Triatoma brasiliensis (Hemiptera: Reduviidae) populations.

Isoenzymes, random amplified polymorphic DNA (RAPD), and morphometry were used to compare genetic variability within and between three populations of Triatoma brasiliensis Neiva, 1911 from northeast Brazil. The isoenzyme profiles were identical for the three populations, whereas the complex RAPD patterns allowed clear discrimination between the three. Morphometric analysis, using characters of the head capsule, also showed discrimination between the three populations but only in comparisons of males, not females. The results show considerable genetic heterogeneity in T. brasiliensis, with an indication of geographic structuring possibly resulting from a recent series of domestication events.

Dynamics of thermopreference in the Chagas disease vector Panstrongylus megistus (Hemiptera: Reduviidae).

The thermopreference of resting P. megistus (Burmeister, 1834) adults was studied in a temperature gradient. We also determined the thermopreference for oviposition and ecdysis. Thermopreference of resting individuals depended on the feeding state of the insects. Results demonstrated an initial resting preference of approximately 290 degrees C, which gradually changed to 26-27 degrees C with increasing starvation. A daily variation in thermopreference was also observed with this species. The insects showed a daily maximum preference at the beginning of the dark phase, and a minimum preference during the first half of the light phase. Ecdysis took place all along the gradient, but exhibited a maximum frequency at 30 degrees C. Most eggs were found between 25 and 29 degrees C. Results are discussed in relation to the ecology of P. megistus as compared with other related species, and the existence of mechanisms of behavioral thermoregulation in the group.

Action of the saliva of Triatoma infestans (Heteroptera: Reduviidae) on sodium channels.

Saliva of Triatoma infestans (Klug) produced a progressive reduction in the amplitude of the compound action potential recorded from rat sciatic nerve. The saliva also inhibited the Na+ current on GH3 cells. The data demonstrate that the saliva of T. infestans has an inhibitory effect on Na+ channels. We conclude that this effect may decrease the generation and conduction of nerve action potential, thereby decreasing the sensitivity of the region in which the insect probes, in a manner similar to that of local anesthetics. This study demonstrates such activity in the saliva of hematophagous insects. The adaptive value of this activity is clear, because the quantity of blood obtained by triatomines is limited by the irritation caused during the feeding process.

The ecology of Triatoma sordida in natural environments in two different regions of the state of Minas Gerais, Brazil.

A study was undertaken about T. sordida in the natural environment in two different regions of the state of Minas Gerais: Itapagipe (Triângulo), an area of cerrado modified by the formation of fields of pasture and agriculture, and Mato Verde (north) an area of transition between caatinga and cerrado with profound deforestation in the last years due to the expansion of cotton cultivation. In both regions the principal ecotopes identified were hollow trees and the bark of live or dead trees, where the occurrence of a food source is not frequent. In this environment, the triatomines utilize various food sources; opposums appear to represent an important source of infection. In the north of Minas, a greater concentration of reservoirs and vectors was observed than in the Triângulo which could explain the higher level of infection of the triatomines in the north. Close attention to the process of domiciliation of T. sordida in the north of Minas is recommended where an extensive intervention by man in the natural environment has occurred and where a rise in the population of triatomines in the peridomestic environment has been observed in recent years.

[Biological susceptibility of Triatoma sordida and Triatoma infestans to deltamethrin and lambdacyhalothrin under field conditions]. / Suscetibilidade biológica do Triatoma sordida e Triatoma infestans a deltametrina e lambdacyhalotrina em condições de campo.

The biological susceptibility of Triatoma sordida and Triatoma infestans to deltamethrin and lambdacyhalothrin was evaluated in three homogeneous groups of houses in the municipality of Posse, Goiás. Each group of houses was treated according to one of the following protocols: deltamethrin 5% wetted powder at 25 mg a.i./m2; deltamethrin 5% concentrated suspension at 25 mg a.i./m2; and lambdacyhalothrin 10% wetted powder at 30 mg a.i./m2. Monitoring of susceptibility to the insecticides was done after 90, 210, 270 and 360 days after the spraying. Each test consisted of an exposure of 10 third-instar nymphs of each species for every house. The insects were kept in contact with the sprayed wall for a period of 72 hours in plastic containers developed by WHO for studies with adult mosquitos. Thirty days after the exposure the death numbers were scored. Results demonstrate persistent activity of the three insecticides for up to a year after spraying and equal biological susceptibilities for both Triatoma sordida and Triatoma infestans to each formulation.

[Natural parasitism of triatominae eggs by Telenomus fariai Lima, 1927 in the laboratory]. / Parasitismo natural de ovos de triatomíneos por Telenomus fariai Lima, 1927 no laboratório.

In November/89 in the insectary of Centro de Estudos "Emmanuel Dias"/Bambuí-MG a high infestation by the microhymenoptera T. fariai was found. Among the 529 eggs examined from P. megistus and T. vitticeps, 375 (70.9%) of them showed a total of 1045 parasites (91.4% females and 8.6% males). Later on it was found that T. infestans eggs were also parasitised by T. fariai but not those from R. neglectus. The observed parasitoid/egg average was 7.9 in P. megistus; 8.7 in T. vitticeps and 10 in T. infestans. The present report has special importance considering the real possibility of T. fariai infestation due to the access of infested triatomine eggs from field captures inducing great damage to colonies maintained for research purposes. These eggs have to be carefully examined, isolated and eliminated. This procedure and the closing of triatomine containers with thin net (0.25 mm) is show to be efficient in T. fariai control.