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Rhodnius heme-binding protein (RHBP) is a heme source for embryonic development in the blood-sucking bug Rhodnius prolixus (Hemiptera, Reduviidae).

We have previously shown that Rhodnius prolixus' eggs and hemolymph are pink due to the presence of the hemeprotein Rhodnius heme-binding protein (RHBP). In the hemolymph it functions as an antioxidant. Nevertheless, its function in eggs has not been determined. Here we present evidence that RHBP is a source of heme for embryonic development. RHBP content decreases during embryogenesis, but the total heme content of eggs remains unchanged. Biliverdin, the product of heme degradation, is not detectable in late embryos. The activity of the heme-synthesizing pathway is low throughout embryogenesis and rises sharply after nymphs' hatching. Heme-radiolabeled eggs were produced and, at the day of hatching, nymphs were dissected. The presence of radiolabeled heme in their carcass is an indication that heme reutilization is occurring. The only animal known to reutilize heme in significant levels is the cattle tick Boophilus microplus, which cannot synthesize its own heme. Diversely, Rhodnius can synthesize its own heme but, in the context of embryogenesis, heme demand seems to be supplied by the programmed release of heme form RHBP. This behavior indicates that in Rhodnius, we might have a highly unusual profile: heme is both synthesized and reutilized.

Animals↗

Uptake of Rhodnius heme-binding protein (RHBP) by the ovary of Rhodnius prolixus.

The uptake of RHBP (Rhodnius heme-binding protein) by the ovaries of Rhodnius prolixus was characterized. RHBP purified from occyte was labeled with 125I and used to study the process of uptake by the ovary in vivo and in vitro. After injection, the [125I]RHBP was readily removed from the hemolymph and accumulated especially in the ovary. The capacity of the ovary to take up [125I]RHBP from the hemolymph varied during the days following blood meal. It increased up to day 2, remained stable until day 5, and then decreased up to the end of oogenesis. In vitro, the uptake of [125I]RHBP was linear at least up to 60 min. The uptake was dependent on [125I]RHBP concentration and showed to be a saturable process. The addition of a molar excess of non-related proteins such as Vitellin (Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) did not reduce [125I]RHBP uptake. Using immunogold technique the RHBP was localized at the microvilli, coated pits, and yolk granules. The main yolk protein, Vt, did not compete with RHBP for the uptake. Thus, it is discussed here that they bind to independent binding sites of the oocytes, and are directed later on to the same compartment. The need of both proteins for the completion of mature oocyte was verified in vivo. The reduction of heme-RHBP in the hemolymph, by changing the diet, decreased the number of eggs laid. Increasing the concentration of heme-RHBP in the hemolymph, the number of eggs produced increased in a dose dependent manner. In vitro, both apo-RHBP and heme-RHBP can be taken up by the oocyte. Since the mature oocyte contains only heme-saturated RHBP, the possible fate of apo-RHBP is also discussed.

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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.

Animals↗

Wing shape divergence between Rhodnius prolixus from Cojedes (Venezuela) and Rhodnius robustus from Mérida (Venezuela).

The existence of Rhodnius robustus as a species distinct from Rhodnius prolixus has long been the main epidemiological question about Chagas disease transmission in Venezuela and surrounding countries. These two taxa are morphologically and genetically very similar, but only R. prolixus is assumed to colonize houses and transmit Chagas disease to humans. R. robustus is assumed to be an exclusively sylvatic species, restricted to palm trees. If robustus and prolixus are actually the same species, the theoretical possibility exists of sylvatic specimens invading houses, even after insecticide application, and a control strategy similar to that of the successful Southern Cone Initiative against Triatoma infestans would be difficult to consider. Since no valid alternative control strategy exists, the answer to this biological question could be decisive about the future of vector control in this region. Although we believe genetic techniques are best suited to define species boundaries, we present here an example of the relevance of modern morphometrics in dealing with such an issue. Using both traditional and geometric morphometrics, we compared the wing size and shape in both sexes of these two taxa reared in the same laboratory for one generation. R. robustus specimens were collected from palm trees in the state of Mérida (Venezuela), and R. prolixus were collected from houses in the state of Cojedes (Venezuela). Our study provided no argument to question their specific status. Even after one generation of living in the same laboratory conditions, the two lines showed clear size differences, divergent allometric trends, and significant allometry-free differences in shape. These results suggest that R. robustus (Mérida, Venezuela) and R. prolixus (Cojedes, Venezuela) are distinct evolutionary units. Due to the epidemiological importance of this question, further studies in other geographic areas of Venezuela are required to accurately define the relationships of R. robustus and R. prolixus.

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Chagas' disease in the Amazon basin: V. Periurban palms as habitats of Rhodnius robustus and Rhodnius pictipes--triatomine vectors of Chagas' disease.

Trypanosoma cruzi infected Rhodnius robustus and/or Rhodnius pictipes were commonly found, in large numbers, in the Brazilian Amazonian palms Maximiliana regia ("inajá"), Acrocomia sclerocarpa ("mucajá") and Orbignya speciosa ("babaçu"). The common opossum, Didelphis marsupialis, was the animal most frequently associated with triatomine infested palms. R. pictipes, frequently light-attracted into houses from palm trees, was the probable source of an acute case of Chagas' disease in the vicinity of Belém. It is considered that triatomine infested palms are likely to cause some cases of acute Chagas' disease in the States of Amazonas and Rondônia. Possible control methods are suggested.

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The role of eicosanoids on Rhodnius heme-binding protein (RHBP) endocytosis by Rhodnius prolixus ovaries.

The participation of eicosanoids and second messengers on the regulation of RHBP endocytosis by the ovaries was investigated, using [(125)I]RHBP in experiments in vivo and in vitro. Addition of PGE(2) (one of the products of the cyclooxygenase pathway) decreased in vitro the uptake of RHBP by 35%. The rate of RHBP endocytosis increased in the presence of indomethacin, a potent cyclooxigenase inhibitor, up to 50% in vitro and up to 55% in vivo, thus giving support to the role of cyclooxygenase derivatives on endocytosis regulation. The amount of PGE(2) secreted to the culture medium by the cells of Rhodnius prolixus ovaries was 1.1 ng/ovary following RHBP uptake assay. The amount of PGE(2) decreases approximately 25% in the presence of 5 microM indomethacin. Using a scanning electron microscope we have observed that neither the surface area nor the patencies of follicle cells were affected by treatment with indomethacin, thus suggesting that, its effect is elicited in the oocyte. Finally, we have identified two ovarian peptides that were dephosphorylated after the indomethacin treatment (18 and 25 kDa). Taken together these data show that local mediators such as eicosanoids act upon the oocytes controlling RHBP endocytosis, perhaps using the protein phosphorylation signal transduction pathway.

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[Susceptibility of Rhodnius neglectus, Rhodnius robustus and Triatoma infestans (Hemiptera, Reduviidae, Triatominae) to infection by two strains of Trypanosoma cruzi (Kinetoplastidae, Trypanosomatidae) using artificial xenodiagnosis].

The susceptibility of 3rd instar nymph of Triatominae Rhodnius neglectus, R. robustus and Triatoma infestans to Trypanosoma cruzi Y and AMJM strains was verified using artificial xenodiagnosis. After the accomplishment of the xenodiagnosis, the faeces of the Triatominae were analyzed on two-day intervals from day 5 until day 31 post infection, using the abdominal compression technique. The results showed differences in the susceptibility of the Triatominae for the two strains studied, and the optimal period reading differed from day 11 to day 19 for the Y strain and from day 11 to day 15 for the AMJM strain. For the Y strain, all three Triatominae species showed good susceptibility, whereas in the AMJM strain, the highest susceptibility was observed with R. neglectus, followed by T. infestans and R. robustus.

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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.

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Changes associated with laboratory rearing in antennal sensilla patterns of Triatoma infestans, Rhodnius prolixus, and Rhodnius pallescens (Hemiptera, Reduviidae, Triatominae).

We examined changes in the array of antennal sensilla of three species of Triatominae (Triatoma infestans, Rhodnius prolixus, and R. pallescens) following their establishment for different periods in laboratory culture. In each case, the laboratory colonies were compared with conspecific samples taken directly from the field, by quantitative analysis of the sensilla arrays on the three distal segments of the antenna in terms of the densities of three types of chemoreceptors (basiconics and thick and thin walled trichoids) and one type of mechanoreceptor (bristles). Sensilla densities were compared by ANOVA or non-parametric tests, and by multivariate discriminant analysis. Strains of the same species reared in different laboratories showed significant differences in their sensilla arrays, especially when compared to field-collected material from the same geographic origin. A Bolivian strain of T. infestans reared in the laboratory for 15 years and fed at monthly intervals, showed greatest differences from its conspecific wild forms, especially in terms of reductions in the number of chemoreceptors. By contrast, an Argentine strain of T. infestans reared for 25 years in the laboratory and fed weekly, showed a relative increase in the density of mechanoreceptors. A Colombian strain of R. prolixus reared for 20 years and fed weekly or fortnightly, showed only modest differences in the sensilla array when compared to its wild populations from the same area. However, a Colombian strain of R. pallescens reared for 12 years and fed fortnightly, did show highly significant reductions in one form of chemoreceptor compared to its conspecific wild populations. For all populations, multivariate analysis clearly discriminated between laboratory and field collected specimens, suggesting that artificial rearing can lead to modifications in the sensory array. This not only supports the idea of morphological plasticity in these species, but also suggests caution in the use of long-established laboratory material for experimental studies designed to extrapolate the natural behaviour and physiology of these species.

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Ecdysteroidogenic action of Bombyx prothoracicotropic hormone and bombyxin on the prothoracic glands of Rhodnius prolixus in vitro.

An in-vitro assay for ecdysteroid synthesis by the prothoracic glands (PGs) of fifth instar Rhodnius prolixus has been employed to evaluate the actions of prothoracicotropic neuropeptides from the silkmoth, Bombyx mori. Crude prothoracicotropic hormone (PTTH) extracts of recently emerged adult brain complexes of Bombyx induced a dose-dependent stimulation of ecdysteroid synthesis by Rhodnius PGs, which was similar to that obtained using crude Rhodnius PTTH. In both cases, maximum stimulation was obtained with one brain equivalent. Rhodnius PGs were then challenged with incremental doses of recombinant Bombyx PTTH and synthetic bombyxin-II. Dose-response curves for the action of both peptides on Rhodnius PGs were very similar to those obtained for their action on the pupal PGs of Bombyx in vitro. Bombyx PTTH stimulated the PGs of Rhodnius at concentrations comparable to those effective on Bombyx. The curve for Bombyx PTTH showed a steep ascending region from 3 to 8ng/ml and a sharp peak. For bombyxin, concentrations 40-fold higher were required to elicit the same amount of stimulation as obtained using Bombyx PTTH. Therefore, Rhodnius PGs possess recognition sites for both Bombyx PTTH and bombyxin. This is the first study of the ecdysteroidogenic properties of the Bombyx peptides on a heterologous species. It is suggested that the function and conformation of PTTH may be conserved between distantly related insect groups.

Journal Article↗

Molecular characterisation of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis in Peru, R. colombiensis in Colombia and R. pallescens in Panama, supports a co-evolutionary association between parasites and vectors.

We present data on the molecular characterisation of strains of Trypanosoma rangeli isolated from naturally infected Rhodnius ecuadoriensis in Peru, from Rhodnius colombiensis, Rhodnius pallescens and Rhodnius prolixus in Colombia, and from Rhodnius pallescens in Panama. Strain characterisation involved a duplex PCR with S35/S36/KP1L primers. Mini-exon gene analysis was also carried out using TrINT-1/TrINT-2 oligonucleotides. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: (i) KP1, KP2 and KP3 or (ii) KP2 and KP3 products for kDNA, and 380 bp or 340 bp products for the mini-exon. All T. rangeli strains isolated from R. prolixus presented KP1, KP2 and KP3 products with the 340 bp mini-exon product. By contrast, all T. rangeli strains isolated from R. ecuadoriensis, R. pallescens and R. colombiensis, presented profiles with KP2 and KP3 kDNA products and the 380 bp mini-exon product. Combined with other studies, these results provide evidence of co-evolution of T. rangeli strains associated with different Rhodnius species groups east and west of the Andean mountains.

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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.

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Differential susceptibility of triatomines of the genus Rhodnius to Trypanosoma rangeli strains from different geographical origins.

The susceptibility of four Rhodnius species to different Trypanosoma rangeli strains was evaluated using both intracoelomic inoculation and oral infection. Rhodnius prolixus, Rhodnius domesticus, Rhodnius neglectus and Rhodnius nasutus were infected with Trypanosoma rangeli Macias (Venezuela), Choachi (Colombia) and SC-58 (Brazil) strains, revealing distinct haemolymph and salivary glands infection rates. The obtained infection rates were revealed to be dependent on the method of infection and the triatomine species. Our results suggest the existence of a high adaptation between the strain and the local vector.

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The distribution of a kinin-like peptide and its co-localization with a CRF-like peptide in the blood-feeding bug, Rhodnius prolixus.

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay. In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s). The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.

Animals↗

Serotonin: a coordinator of feeding-related physiological events in the blood-gorging bug, Rhodnius prolixus.

Rhodnius prolixus is an obligatory blood-feeder that can ingest blood meals of up to 10 times its mass. Rapid production of urine commences within 2-3 min of the start of feeding in order to eliminate the load of water and salts, and so there is an increase of Malpighian tubule secretion greater than 1,000 fold in response to feeding. Feeding and post-prandial diuresis in Rhodnius are highly coordinated events, including for example, host recognition, probing, injection of saliva, cuticle plasticization, passage of blood through the digestive system, diuresis and excretion. This review illustrates that many of the known functions of serotonin in Rhodnius are feeding-related. Serotonin coordinates or 'orchestrates' feeding-related physiological events either as a neurotransmitter/neuromodulator, delivered to target tissues in the nerve supply, or as a neurohormone, delivered by the haemolymph. Thus, serotonin has physiological effects upon the salivary glands, cuticle, digestive tract, cardiac muscle, and Malpighian tubules. By discussing these aspects, the review illustrates that serotonin acts in a coordinated manner to prepare Rhodnius for this energy-demanding process of feeding and diuresis.

Animals↗

The distribution and effects of Dippu-allatostatin-like peptides in the blood-feeding bug, Rhodnius prolixus.

Using a polyclonal antiserum to Dippu-allatostatin 7 (Dippu-AST 7; formerly AST 1) of the cockroach Diploptera punctata, we have demonstrated the presence of AST-like immunoreactivity (ALI) in cells and processes throughout the nervous system, gut, and peripheral tissues of unfed fifth instar and adult Rhodnius prolixus. ALI in apparent neurosecretory cells of the brain, suboesophageal ganglion, and mesothoracic ganglionic mass, as well as in midgut endocrine cells, suggests that Rhodnius allatostatins may act as neurohormones/hormones. The presence of ALI in possible interneurons and areas of neuropile throughout the CNS also suggests roles as neuromodulators and/or neurotransmitters. Dippu-AST 7 inhibits spontaneous and leucokinin 1 (LK 1)-induced contractions of the Rhodnius hindgut in a dose-dependent manner. The low concentrations capable of inhibiting both spontaneous (10(-12)M) and LK 1-induced contractions (10(-10) to 10(-9)M) suggest that ASTs may be acting as neurohormones/hormones on the hindgut. We have also shown that Dippu-AST 7 influences the muscle activity of the Rhodnius dorsal vessel at concentrations as low as 10(-11)M.

Abdomen↗