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.

Head morphometry and isoenzymatic profile of two Triatoma infestans Klug, 1834 (Hemiptera, Reduviidae) populations.

The triatomine bug Triatoma infestans was probably originated in Bolivia and dispersed passively over large areas of South America, where it is the major vector of Trypanosoma cruzi. In its probable origin area this species shows two different patterns of behaviour, being found both in sylvatic and human related habitats. Such behaviour is not observed in other areas of its distribution, where it is exclusive to human related habitats. The main objective of the present study was to evaluate head morphometry characters and isoenzymes patterns of two T. infestans populations, one collected in Minas Gerais (Brazil) and other in the Cochabamba Valley (Bolivia), in order to elucidate the factors that could have an implication with the different colonization behaviour. The two populations presented differences in both morphometric (head size) and isoenzymes (specially PGM enzyme profile). The insects from the colonies reared for several years in laboratory conditions, showed reduction in head size starting from the third generation, maintaining this same size in all generations after, until 10 years after generation. The laboratory rearing conditions could be similar to human associated habitats, inducing certain level of genetic homogeneity. Our results showed increase of genetic homogeneity in Brazilian and laboratory-reared colonies from Bolivia, that may be due to a selection process due to passive dispersion, followed by several founder effects episodes and genetic drift.

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.

New perspectives for population genetics of Chagas'disease vectors in the Northeastern Brazil: isolation of polymorphic microsatellite markers in Triatoma brasiliensis.

Triatoma brasiliensis is the most important Chagas disease vector in semiarid areas of Northeastern Brazil. Although generally found in natural environment, it regularly colonizes or reinvades domiciliary ecotopes. In order to analyse gene flow between habitats, we identified and characterized six microsatellite loci using a microsatellite-enriched genomic library. To assess the usefulness of these microsatellites for genetic studies, we investigated their variability in two natural populations of the T. brasiliensis complex sampled in two Brazilian districts (Corone Jose Dia, South of Piaui state; Curaça, North of Bahia state). The taxonomic status of the samples was checked using cytb sequences. Phylogenetic analyses confirmed that the individuals from Bahia belonged to the T. juazeirensis species. Moreover, primers cross-amplification was tested in 5 Triatoma species and 4 loci successfully amplified in T. infestans and T. guasayana.

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.

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).

Molecular cloning and sequencing of salivary gland-specific cDNAs of the blood-sucking bug Triatoma brasiliensis (Hemiptera: Reduviidae).

Haematophagous insects produce pharmacological substances in their saliva to counteract vertebrate host haemostasis events such as coagulation, vasoconstriction and platelet aggregation. To investigate the bioactive salivary molecules of the triatomine bug Triatoma brasiliensis, we produced subtraction-enriched cDNAs of salivary-gland specific genes using suppression subtractive hybridization. Six full-length differentially expressed cDNAs (Tb113, Tb125, Tb152, Tb169, Tb180 and Tb198) were selected, cloned and sequenced. Sequence similarity searches of the databases using the putative amino acid sequence of our clones gave the following results: Tb152 - Triabin, an antithrombin induced platelet aggregation factor found in salivary gland extracts of T. pallidipennis. Tb169 - Pallidipin, an anticollagen induced platelet aggregation factor also found in T. pallidipennis salivary homogenates. Tb180 - Procalin, the major allergen of T. protracta saliva. The other three salivary-gland specific cDNAs produced no obvious homologies. Comparison of these salivary gland-specific cDNAs of with those of other triatomines combined with functional studies using recombinant proteins will allow a better understanding of the co-evolutionary process occurring between these insects and their vertebrate hosts, and may also lead to the discovery of novel antihaemostatic agents.

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.

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.

Experimental evidence for a demographic cline in Panstrongylus megistus populations.

The population biology of three populations of Panstrongylus megistus was compared to determine possible influence on the behaviour and epidemiological importance of this species. The results demonstrated differences in terms of egg eclosion time, nymphal mortality and development rates, and feeding and defaecation rates. These differences appeared to follow a geographical cline, primarily reflecting different degrees of adaptation to domestic habitats.

Genetic studies of Psammolestes tertius (hemiptera: reduviidae: triatominae) using male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA.

Two Brazilian populations of Psammolestes tertius (Ceará and Minas Gerais) collected from thornbird nests (Furnariidae) were compared by male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA (RAPD). Male genitalia showed no difference between the populations. In contrast, morphometry, isoenzyme, and RAPD clearly distinguished the two populations. Possible mechanisms of dispersal and the origin of Psammolestes are discussed.

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.

[Operational aspects of Triatoma brasiliensis control]. / Aspectos operacionais do controle do Triatoma brasiliensis.

Vector control strategies against indigenous species is not easy, due to their capacity to reinvade treated premises from sylvatic ecotopes. Between August 1996 and December 1997 we conducted a study on reinfestation of houses after spraying in a county in the State of Ceará. Of 277 houses examined, 113 (40.8%) were infested (21.7% intradomiciliary and 35.4% peridomiciliary). Of the 433 Triatominae collected, 207 were Triatoma brasiliensis (49% of which intradomiciliary, with a mean of 1.8 insects/house) and 226 were Triatoma pseudomaculata (97% peridomiciliary). The age structure of the two indicated a univoltine development cycle for T. brasiliensis and two cycles per year for T. pseudomaculata. Four months after spraying with deltamethrin SC 25mg ia/m2, 9.7% of the houses were still positive, mainly with peridomestic infestations. Intradomiciliary wall bioassays showed persistence of the insecticide up to 9 months after spraying. Considering the high potential for recolonization of treated premises from sylvatic foci, we propose an operational strategy combining traditional evaluations and community-based surveillance with increased selective interventions and community education.

[Microclimatic properties of the Triatoma brasiliensis habitat]. / Aspectos microclimáticos del hábitat de Triatoma brasiliensis.

Vector-borne transmission of Chagas disease in Northeast Brazil is basically by Triatoma brasiliensis. It is thus crucial to determine this species' microclimatic preferences as limiting factors for its distribution and ability to infest domestic environments. We analyze the microclimatic properties of the shelters in which these insects are found in wild, domestic, and peridomiciliary environments in the State of Ceará, at Brazil. We measure temperature and relative humidity (RH) every 15 minutes for 3 days. Thermal variation was greatly dampened inside both domiciliary refuges and the more protected internal places in wild stony sites. For RH, we observed a similar dampening pattern, but mean RH was lower in both domiciliary refuges and wild ones inside stony sites as compared to reference levels in the surrounding environment. The results are discussed with regard to this species' microclimatic preferences in the laboratory and its potential as determinants of its geographical distribution.

Chromosome homogeneity in populations of Triatoma brasiliensis Neiva 1911 (Hemiptera - reduviidae - triatominae).

Triatoma brasiliensis is the most important vector of Chagas disease in the semiarid zone of the Northeast of Brazil. Several authors have reported the occurrence of four chromatic patterns with morphological, ecological, and genetic differences. In order to determine the existence of cytogenetic differentiation between these chromatic forms, we analyzed their karyotypes and the chromosome behavior during the male meiotic process. Triatoma brasiliensis shows distinct and specific chromosome characteristics, which differ from those observed in all other triatomine species. However, no cytogenetic differences were observed between the four chromatic forms of T. brasiliensis. The lack of chromosome differentiation among them could indicate that the populations of this species are in a process of differentiation that does not involve their chromosomal organization.

[Use of random amplified polymorphic DNA (RAPD) in the populational study of Triatoma brasiliensis Neiva, 1911]. / Uso do Random Amplified Polymorphic DNA (RAPD) no estudo populacional do Triatoma brasiliensis Neiva, 1911.

We evaluated the genetic variability of Triatoma brasiliensis, the main vector of Chagas disease in Northeast Brazil, using specimens from three populations. Regions of genomic DNA were amplified by RAPD (Random Amplified Polimorphic DNA), using two primers. The products were visualized after polyacrylamide gel electrophoresis followed by silver staining. A dendrogram constructed through the Dice similarity coefficient allowed for separation of the tested specimens into three distinct groups. The populations captured in areas from Ceará State showed similar profiles, but different from that captured in Piauí State. Our results indicate that RAPD can be used successfully in triatomine studies and suggest the presence of genetic variability between different populations of T. brasiliensis.

[Biological potential of Triatoma brasiliensis]. / Potencial biológico do Triatoma brasiliensis.

Biological and physiological parameters of Triatoma brasiliensis, Triatoma infestans, and Triatoma pseudomaculata were studied and compared. T. brasiliensis, reared on mice, showed a faster evolutionary cycle from first stage to adult and higher fecundity, when compared to the other species. T. infestans showed the fastest blood intake in both hosts tested, followed by T. brasiliensis and T. pseudomaculata. Clotting tests using salivary gland extracts of T. brasiliensis presented intermediary values of anti-clotting activity when compared to T. infestans and T. pseudomaculata.

Comparative developmental and susceptibility to insecticide of Bolivian and Brazilian populations of Triatoma infestans.

The triatomine bug Triatoma infestans probably originated in Bolivia and dispersed passively over wide areas of South America, where it is the principal vector of Trypanosoma cruzi. In the region of its probable origin this species shows colonization in two different ecotopes, so that it may be encountered in sylvatic as well as in artificial habitats. The sylvatic colonization pattern is not observed in the rest of its range, where T. infestans is exclusive to man-made habitats. The objective of this study was to compare several aspects of two T. infestans populations, one from Minas Gerais (Brazil) and the other from the Cochabamba Valley (Bolivia), with a view to elucidate the factors associated with the different colonization patterns observed for this species. The differences between the developmental cycle, weight, capacity to ingest blood and mortality rate of first instar nymphs should indicate more fragility of Brazilian population that may be related to its elimination possibility.