Allozyme relationships among ten species of Rhodniini, showing paraphyly of Rhodnius including Psammolestes

Genetic relationships among 10 species of bugs belonging to the tribe Rhodniini (Hemiptera: Reduviidae), including some important vectors of Chagas disease, were inferred from allozyme analysis of 12 enzyme loci (out of 21 enzyme systems examined), using agarose gel electrophoresis. These species formed two clusters: one comprising Rhodnius brethesi, R. ecuadoriensis, R. pallescens and R. pictipes; the other with Psammolestes tertius, Rhodnius domesticus and the Rhodnius prolixus group comprising R. nasutus, R. neglectus, R. prolixus and R. robustus. The resulting tree was [((R. ecuadoriensis, R. pallescens) R. brethesi) R. pictipes], [R. domesticus (P. tertius [(R. nasutus, R. neglectus) (R. prolixus, R. robustus)])]. Rhodnius nasutus and R. neglectus differed by only one locus, whereas no diagnostic loci were detected between R. prolixus and R. robustus (22 loci were analysed for these four species), despite considerable DNA sequence divergence between species in each of these pairs. Allozymes of the R. prolixus group showed greater similarity with Psammolestes tertius than with other Rhodnius spp., indicating that Rhodnius is paraphyletic and might include Psammolestes.     

Feeding and defecation of Rhodnius (hemiptera: Reduviidae) fed human blood

Feeding and defecation behavior of Rhodnius prolixus Stal, 1859, R. robustus Larrousse, 1927, R. neivai Lent, 1953 and R. pictipes Stal, 1872, artificially fed on human blood, were studied under laboratory conditions. In all species, first instar nymphs did not defecate in the first 30 minutes after feeding. R. pictipes did not accept artificial feeding but fed directly on humans. Nymph and adult R. prolixus had a higher defecation index (DI) than other species; third instar nymphs had the highest DI = 1.62. In all instars, most individuals accepted the food in 3 Pounds minutes and finished feeding in less than 15 minutes.     

Rhodnius robustus in Bolivia identified by its wings.

Wings of a Rhodnius specimen from Alto Beni (Bolivia) was examined for identification and compared with R. stali, R. robustus, (certified Bolivian species), R. pictipes and R. prolixus (suspected Bolivian species). A projection of the unidentified wings as supplementary data into a discriminant analysis of shape revealed clear cut differences with R. stali and R. pictipes, less differences with R. prolixus, and none with R. robustus. Combining global size and shape of the wings, the unknown specimen was identified as R. robustus. Thus, this study confirmed the presence of R. robustus in Bolivia. It also highlighted the possibility of morphometrics to taxonomically interpret one individual, or even one piece of an individual, when related species data are available for comparison.     

Esterase patterns in species of the triatome bug Rhodnius

The aim of the present study was to analyse esterase patterns in three triatomine species of Rhodnius genus. Four loci, Est 1, Est 2, Est 3 and Est 4, were found. The corresponding enzymes were characterized as carboxylesterases (E.C. 3.1.1.1) or cholinesterases (E.C. 3.1.1.8) based on inhibitory experiments, using eserine sulphate, malathion, mercury chloride, p-chloromercuribenzoate (pCMB) and iodoacetamide. Low genetic variability was observed: Est 1, Est 2 and Est 3 were monomorphic in Rhodnius domesticus , Rhodnius robustus and Rhodnius neivai , whereas locus Est 4 was polymorphic in the first two species. The UPGMA analysis based on esterase genotypic frequencies indicated greater similarity between R. domesticus and R. robustus when compared with R. neivai . The present study expands our knowledge about genetic variability among triatomines and accords with the hypothesis that R. domesticus is a species derived from R. robustus .     

Molecular phylogeography of the Amazonian Chagas disease vectors Rhodnius prolixus and R. robustus

The phylogeographical structure of the closely related species Rhodnius prolixus and R. robustus is presented based on a 663-base pair (bp) fragment of the mitochondrial cytochrome b gene. Twenty haplotypes were recovered from 84 samples examined, representing 26 populations from seven Latin American countries. The resulting phylogenetic tree is composed of five major reciprocally monophyletic clades, one representing R. prolixus and four representing R. robustus. While R. prolixus is a very homogeneous assemblage, R. robustus has deeper clades and is paraphyletic, with the clade comprising R. robustus from Venezuela (Orinoco region) more closely related to the R. prolixus clade than to the other R. robustus populations from the Amazon region. The R. robustus paraphyly was supported further by the analysis of a nuclear gene (D2 region of the 28S RNA) for a subset of specimens. The data support the view that R. robustus represents a species complex. Levels of sequence divergence between clades within each region are compatible with a Pleistocene origin. Nucleotide diversity (pi) for all R. prolixus populations was extremely low (0.0008), suggesting that this species went through a recent bottleneck, and was subsequently dispersed by man.     

Studies on Trypanosoma rangeli Tejera, 1920. VII--Its effect on the survival of infected triatomine bugs

The pathological effects of Trypanosoma rangeli on Rhodnius prolixus and R. robustus, and the relation of mortality to infection, were studied under laboratory conditions. Frequent observations revealed that when the first instar nymphs of R. prolixus and R. robustus were infected with T. rangeli, survival of the bugs during the stages of development to the adult stage decreased. This decrease was statistically significant when compared with uninfected control-bugs, indicating that T. rangeli is pathogenic for both species of triatomine. In R. prolixus the most affected nymphal stages were the first, second and fifth instars, where a higher mortality was also observed. In R. robustus a progressive increase of the mortality from the first to fifth instars, was observed. The pathogenicity of T. rangeli as measured by overall mortality was the same in R. prolixus and R. robustus. The possible pathogenic mechanism of T. rangeli in triatomine-bugs and its epidemiological implications, are discussed.     

Cytogenetics as a tool for triatomine species distinction (Hemiptera-Reduviidae).

Several cytogenetic traits were tested as species diagnostic characters on five triatomine species: Rhodnius pictipes, R. nasutus, R. robustus, Triatoma matogrossensis and T. pseudomaculata. Four of them are described for the first time. The detailed analysis of the meiotic process and the application of C-banding allowed us to identify seven cytogenetic characters which result useful to characterize and differentiate triatomine species.     

Studies on vectors of Trypanosoma cruzi in Trinidad, West Indies

Studies on vectors of Trypanosoma cruzi Chagas were conducted during 1982-85 as part of an investigation on the epidemiological status of American Trypanosomiasis in Trinidad, West Indies. A total of 148 reduviid bugs were collected from caves, armadillo nests and at light traps, comprising four sylvatic species and totalling 120 Panstrongylus geniculatus (Latreille), two P. rufotuberculatus (Champion), twelve Rhodnius pictipes Stal and fourteen Eratyrus mucronatus Stal. A significantly higher level of infection with T. cruzi was recorded for P. geniculatus (42.5%) compared to R. pictipes (8.3%). The other two bug species were not found to be infected. P. geniculatus seems to be the most important vector in the sylvatic cycle of transmission of T. cruzi in Trinidad.     

The biogeography and population genetics of neotropical vector species

Phylogenetic and population genetic data support the Pliocene or Pleistocene divergences of the co-distributed hematophagous insect vectors, the sand fly Lutzomyia longipalpis s.l., the mosquitoes Anopheles darlingi and A. albitarsis s.l., and the triatomines Rhodnius prolixus and R. robustus. We examined patterns of divergence and distribution in relation to three hypotheses of neotropical diversification: Miocene/Pliocene marine incursion, Pliocene/Pleistocene riverine barriers and Pleistocene refugia. Only R. prolixus has a pattern concordant with the refugia hypothesis, and R. robustus conforms to the marine incursion predictions. A. darlingi partially fits the refugia hypothesis. For L. longipalpis s.l. and A. albitarsis s.l., elements of both incursion and refugia hypotheses seem to fit, suggesting perhaps an interaction of factors determining their distribution patterns.     

Polymorphism of the ITS-2 region of the ribosomal DNA of the Triatominae Rhodnius domesticus, R. pictipes, R. prolixus and R. stali

Abstract The polymerase chain reaction?restriction fragment length polymorphism technique (PCR?RFLP) was used to compare Rhodnius domesticus (Neiva & Pinto), R. pictipes (Stal), R. prolixus (Stal) and R. stali (Lent, Jurberg & Galvão) (Hemiptera: Reduviidae). The enzyme BstUI differentiated R. domesticus, R. pictipes and R. prolixus, and HhaI differentiated R. domesticus, R. pictipes and R. stali. With the fingerprinting analysis generated by these two enzymes, it was possible to clearly identify all four species in the study.     

Epidemiology of Chagas disease in Guatemala: infection rate of Triatoma dimidiata,Triatoma nitida and Rhodnius prolixus (Hemiptera,Reduviidae) with Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida,Trypanosomatidae)

A five-year domiciliary collection in the 22 departments of Guatemala showed that out of 4,128 triatomines collected, 1,675 were Triatoma dimidiata (Latreille, 1811), 2,344 were Rhodnius prolixus Stal 1859, and only 109 were T. nitida Usinger 1939. The Chagas disease parasite, Trypanosoma cruzi, was found in all three species. Their natural infection rates were similar in the first two species (20.6%; 19.1%) and slightly lower in T. nitida(13.8%). However there was no significant difference in the infection rates in the three species (p = 0.131). T. dimidiata males have higher infection rates than females (p = 0.030), whereas for R. prolixus there is no difference in infection rates between males and females (p = 0.114). The sex ratios for all three species were significantly skewed. More males than females were found inside houses for T. dimidiata (p < 0.0001) and T. nitida (p = 0.011); a different pattern was seen for R. prolixus (p = 0.037) where more females were found. Sex ratio is proposed as an index to show the mobility of T. dimidiata in different populations. T. dimidiata is widely distributed in the country, and is also the main vector in at least ten departments, but R. prolixus with higher vectorial capacity is an important vector in at least two departments.     

Feeding behaviour of morphologically similar Rhodnius species: influence of mechanical characteristics and salivary function

Despite their morphological similarities, very similar Rhodnius species (R. prolixus, R. robustus, R. nasutus and R. neglectus) displayed a distinct feeding behaviour when fed on artificial feeder, pigeon or mouse. On pigeon hosts, these species showed distinct groups in terms of cumulative probing time - quicker species (R. prolixus and R. neglectus) followed by R. nasutus and finally a much slower species (R. robustus). On mouse hosts, R. nasutus showed quicker probing time compared to the other three species. Moreover, R. prolixus displayed quicker probing time compared to R. robustus and R. neglectus. Except for R. nasutus, the mean total ingestion rate tended to have different values between feeding sources (artificial feeder>pigeon>mouse). The volume ingested by each cibarial pump contraction and maximum frequency obtained using the artificial feeder are expected to be related to intrinsic mechanical characteristics of the insect feeding apparatus. However, probing time and the modulation of cibarial pump frequency on live hosts may be related to salivary function. R. prolixus showed high mechanical and salivary efficiency, achieving high values of total ingestion rate when fed on artificial feeder or either of the hosts. Comparative analysis suggests that species which possess higher total ingestion rates tend to achieve higher nutritional status, allowing them to reach higher densities.     

Characterization of Rhodnius neglectus from two regions of Brazil using isoenzymes, genitalia morphology and morphometry

Among the triatomines considered as secondary in the epidemiology of Chagas disease, Rhodnius neglectus is frequently captured in artificial ecotopes, especially peridomiciliary ones, rarely producing colonies indoors. Nevertheless, the presence of breeding colonies in houses was unquestionably demonstrated in some areas of the State of Goiás, Brazil. Previous isoenzyme comparisons of this species with morphologically close triatomines, such as R. prolixus, R. robustus or R. nasutus, did not produce definitive conclusions because of doubt about the geographical origin of the R. neglectus. We present here, for the first time, the isoenzyme profile of topotypes of R. neglectus. In addition, wild caught specimens from the type locality, Uberaba (Minas Gerais, Brazil), were compared to wild caught specimens from Jaraguá (Goiás, Brazil), where R. neglectus is more frequently reported invading houses. We used isoenzyme, morphology and morphometry analysis. Neither morphological nor enzymatic differences were found between areas, but metric, size-related divergence was evidenced between them.     

Aspects of classification of Hemiptera hemocytes from six triatomine species.

The objective of this work was to characterize, and compare different morphological types of hemocytes of Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, Triatoma infestans, Panstrongylus megistus, and Dipetalogaster maximus. This information provides the basis for studying the cellular immune systems of these insects. Seven morphological hemocyte types were identified by phase-contrast microscopy: prohemocytes, plasmatocytes, granular cells, cystocytes, oenocytoids, adipohemocytes and giant cells. All seven types of hemocytes are not present in every species. For example, adipohemocytes and oenocytoids were not observed in P. megistus and P. infestans, and giant cells were rarely found in any of the species studied. The hemocytes of Rhodnius and Dipetalogaster are more similar to each other than those from Triatoma and Panstronglus which in turn closely resemble each other. Emphasis is placed on methodological problems arising in this work which are discussed in detail.     

Rhodnius amazonicus Almeida, Santos & Sposina, 1973, bona species, close to R. pictipes Stål, 1872 (Heteroptera, Reduviidae, Triatominae)

The discovery of two atypical specimens of Rhodnius pictipes St?l, 1872 in French Guiana and the examination of the female holotype of R. amazonicus Almeida, Santos & Sposina, 1973, the only specimen of this species so far known, lead us to propose the rehabilitation of R. amazonicus synonymized with R. pictipes. The male is described for the first time and the female redescribed. Both external characters and genitalia distinguish R. amazonicus from R. pictipes. R. amazonicus shows affinities not only with pictipes but also with R. stali Lent, Jurberg & Galv?o, 1993 and R. paraensis Sherlock, Guitton & Miles, 1977. A key is provided for these four species forming, in all likelihood, a natural group, i.e. the "pictipes group".     

Antiserum against perimicrovillar membranes and midgut tissue reduces the development of Trypanosoma cruzi in the insect vector, Rhodnius prolixus

Antiserum raised against Rhodnius prolixus perimicrovillar membranes (PMM) and midgut tissue interfered with the midgut structural organization and reduced the development of Trypanosoma cruzi in the R. prolixus insect vector. SDS-PAGE and Western blot analyses confirmed the specific recognition of midgut proteins by the antibody. Feeding, mortality, molt, and oviposition of the insects were unaffected by feeding with the antiserum. However, the eclosion of the eggs were reduced from R. prolixus females treated with antiserum. Additionally, in vivo evaluation showed that after oral treatment with the antiserum, the intensity of infection with the Dm-28c clone of T. cruzi decreased in the digestive tract of fifth-instar nymphs and in the excretions of R. prolixus adults. These results suggest that the changes observed in the PMM organization in the posterior midgut of R. prolixus may not be important for triatomine survival but the antiserum acts as a transmission-reduction vaccine able to induce significant decreases in T. cruzi infection in the vector.     

Proteolytic activation of canatoxin, a plant toxic protein, by insect cathepsin-like enzymes

Canatoxin is a protein isolated from jackbean (Canavalia ensiformis), seeds. Injected intraperitoneally, the toxin is lethal to mice but it is inactive if given orally. Canatoxin is also lethal when fed to insects with cathepsin-based digestion while insects with trypsin-based digestion are not affected. The hypothesis that canatoxin is proteolytically activated by cathepsins was investigated. Experiments were performed with 4(th) instar and adult Rhodnius prolixus fed meals containing canatoxin (2.5 microg/mg weight body). While 100% of nymphs died, no effect was observed in adults. Hemolymph taken from nymphs and adults showed the presence of canatoxin's proteolytic fragments. Reduced lethality was seen in R. prolixus 4(th) instars fed meals containing canatoxin and inhibitors of cathepsin enzymes, E-64 (2.0 microM) or Pepstatin-A (2. 0 microM). In another approach, canatoxin was digested in vitro with enzymes from the bruchid, Callosobruchus maculatus, and the resulting peptides were tested in R. prolixus. Three groups of toxic peptides (8,000-12,000 kD range) were separated by gel-filtration. When these peptides were fed to the insects simultaneously with the cathepsin inhibitors, no protective effect was seen. These results confirm the proteolytic activation of canatoxin by insect cathepsin-like enzymes to produce entomotoxic peptide(s). Furthermore, our data point towards overlooked differences in the digestive physiology of distinct life stages of R. prolixus. Arch.     

Differentiation and genetic analysis of Rhodnius prolixus and Rhodnius colombiensis by rDNA and RAPD amplification.

Domiciliated Rhodnius prolixus and sylvatic R. colombiensis were analyzed in order to confirm their genetic divergence and verify the risk that the latter represents in the domiciliation process, and to provide tools for identifying the sources of possible reinfestation by triatomines in human dwellings allowing control programs to be undertaken. Comparison of random amplified polymorphic DNA amplification patterns and cluster analysis suggests reproductive discontinuity between the two species. The calculated statistical F value of 0.24 and effective migration rate of 0.6 individuals per generation are insufficient to maintain genetic homogeneity between them and confirm the absence of present genetic flow. R. colombiensis presents higher intrapopulation variability. Polymerase chain reaction of ribosomal DNA supports these findings. The low genetic flow between the two species implies that R. colombiensis do not represent an epidemiological risk for the domiciliary transmission of Trypanosoma cruzi in the Tolima Department. The lower variability of the domiciliated R. prolixus could result in greater susceptibility to the use of pesticides in control programs.     

Attraction of Chagas disease vectors (Triatominae) to artificial light sources in the canopy of primary Amazon rainforest

Adult triatomines occasionally fly into artificially lit premises in Amazonia. This can result in Trypanosoma cruzi transmission to humans either by direct contact or via foodstuff contamination, but the frequency of such behaviour has not been quantified. To address this issue, a light-trap was set 45 m above ground in primary rainforest near Manaus, state of Amazonas, Brazil and operated monthly for three consecutive nights over the course of one year (432 trap-hours). The most commonly caught reduviids were triatomines, including 38 Panstrongylus geniculatus, nine Panstrongylus lignarius, three Panstrongylus rufotuberculatus, five Rhodnius robustus, two Rhodnius pictipes, one Rhodnius amazonicus and 17 Eratyrus mucronatus. Males were collected more frequently than females. The only month without any catches was May. Attraction of most of the known local T. cruzi vectors to artificial light sources is common and year-round in the Amazon rainforest, implying that they may often invade premises built near forest edges and thus become involved in disease transmission. Consequently, effective Chagas disease prevention in Amazonia will require integrating entomological surveillance with the currently used epidemiological surveillance.     

Effect of human blood feeding on the fecundity, fertility and biological cycle of Rhodnius prolixus (Hemiptera: Reduviidae)

The effect of several human blood fractions artificially fed to Rhodnius prolixus Stal 1859 on oviposture (fecundity), egg-hatching (fertility) and life cycle was observed. Specimens fed on man's blood were more fecund than those fed with woman's blood. There were no significant differences in fertility related to host sex. The nymphal development time and number of feedings to molt to the following instar were estimated. Animals fed only on blood plasm did not finish nymphal development, while those fed only blood red cells ended their life cycle in the third nymphal instar. Total life cycle lasts 129 days in individuals fed with whole blood.