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.     

Studies on Trypanosoma rangeli Tejera, 1920. IX. Course of infection in different stages of Rhodnius prolixus

Frequent individual observations od different stages of Rhodnius prolixus exposed to Trypanosoma rangeli, revealed a higher susceptibility to infection in the bugs exposed during the two first instars. The mortality rate in infected bugs was significantly higher than in controls, indicating that the parasite was responsible for the majority of deaths. An analysis of the mortality distribution, per instar, is presented. Statistical analysis of deaths among the different infected instars, showed that T. rangeli produces its pathological effect in any stage of R. prolixus independently of its susceptibility to the parasite. The survival to adult decreased in all the infected instar bugs. A significant longer time to reach the adult stage was observed in the infected bugs when compared with controls, excepting for specimens exposed in the third instar. The epidemiological significance of the present results is discussed.     

Experimental transmission of the parasitic flagellates Trypanosoma cruzi and Trypanosoma rangeli between triatomine bugs or mice and captive neotropical bats

Trypanosoma cruzi and Trypanosoma rangeli-like trypanosomes have been found in a variety of neotropical bat species. In this study, bats (Artibeus lituratus, Carollia perspicillata, Desmodus rotundus, Glossophaga soricina, Molossus molossus, Phyllostomus hastatus) were maintained under controlled conditions, and experiments were conducted to determine how they might become infected naturally with trypanosomes. All bats were first screened for existing infections by hemoculture and the examination of blood smears, and only apparently uninfected animals were then used in the experiments. Proof was obtained that the triatomine bug Rhodnius prolixus would readily feed upon some of the bats, and two species became infected after being bitten by bugs infected with T. rangeli. Some bats also became infected by ingesting R. prolixus carrying T. cruzi, or following subcutaneous or intragastic inoculation with fecal suspensions of R. prolixus containing T. cruzi. P. hastatus became infected after ingesting mice carrying T. cruzi. All of the bats studied inhabit roosts that may be occupied by triatomine bugs and, with the exception of D. rotundus, all also feed to at least some extent upon insects. These findings provide further evidence of how bats may play significant roles in the epidemiology of T. cruzi and T. rangeli in the New World tropics.     

Microclimatic factors influencing refugium suitability for Rhodnius prolixus

Abstract.  Rehydration in the triatomine bug Rhodnius prolixus (Stål) is dependent on the blood meal, and water balance regulation is crucial for survival of starving bugs. In an experimental arena with zones at different climatic conditions, starved R. prolixus nymphs prefer a cooler and more humid zone, stopping there more often and for longer periods. This is probably a behavioural adaptation to limit water loss and reduce metabolic rate. In the Venezuelan State of Portuguesa, temperature and humidity were monitored in three kinds of potential refugia during the dry season: (i) in a palm roof; (ii) in a crack of the wall of a house; and (iii) in a palm tree crown. Fluctuations in temperature and saturation deficit are not very different inside and outside the palm roof except during a few hours of the day when the sun is at its zenith. In the crack of the wall, the diurnal temperature range is reduced to 6.5 °C compared with 12.4 °C outside, and the saturation deficit varies by only 7.6 hPa compared with 28.6 hPa outside. In the palm tree crown, the daily temperature range is only 4.2 °C compared with 13.8 °C outside, and the saturation deficit is permanently < 5 hPa. The microclimatic conditions in the palm tree crown would appear to be ideal for starving R. prolixus , but this kind of refugium generally harbours low densities of bugs, probably related to a combination of predation, pathogens and lower mean temperature within the crown. Such biotic and abiotic constraints play a lesser role in the less ideal palm roof and wall crack refugia where bugs can proliferate as long as hosts are readily available nearby.     

Patterns of expression of odorant receptor genes in a Chagas disease vector

Rhodnius prolixus is a triatomine bug acting as a relevant vector of Chagas disease for which the genome sequence has been recently made available. Based on this information, a set of olfactory (ORs) and ionotropic receptor (IRs) genes potentially related to olfactory processes was characterized, and the expression patterns along bug development and in different structures potentially involved in promoting chemosensory-mediated behaviors were studied. For this, diverse bioinformatic procedures were used to validate gene models analyzing their structural and functional features and designing specific primers. Evolutionary relationships among R. prolixus olfactory coreceptors (RproOrco, RproIR25a, RproIR8a and RproIR76b) and their orthologues from other insects were shown to have mostly good bootstrap support values in phylogenetic trees. Moreover, antennal expression was confirmed for most genes included in the study. Both ORs and IRs showed antennal expression along the whole development of bugs of this species, with few exceptional receptors showing gradually increasing expression or expression restricted to the antennae of adult bugs. Finally, the expression of most of the selected genes was confirmed in other structures, such as rostri, tarsi, tibial pads and genitalia, which are potentially involved in promoting chemosensory-mediated behaviors. These results are discussed in terms of their relevance to advance in the understanding of the molecular bases of triatomine behavior.     

Development of symbionts in triatomine bugs and the effects of infections with trypanosomatids

In the intestinal tract of fifth instars of the hematophagous reduviid bugs Rhodnius prolixus and Triatoma infestans blood ingestion induced an initial decrease of the concentration of the respective symbiotic bacteria Rhodococcus rhodnii and Nocardia sp. and then within 10 days a 15- or 18-fold increase of the total population/bug to about 0.8 x 10(9) colony-forming units in R. prolixus and 1.8 x 10(9) colony-forming units in T. infestans. About 95-99% of the total populations of both symbionts developed in the anterior midgut regions, i.e., cardia and stomach. The passage from the blood-storing stomach to the digesting small intestine caused a considerable breakdown of symbiont populations, and only about 0.01% of the total population was present in the rectum. These were excreted mainly within 4 h after a blood meal. After infection with three species of trypanosomatids, R. rhodnii, the symbiont of R. prolixus, was affected by Trypanosoma rangeli, but not by Blastocrithidia triatomae or Trypanosoma cruzi. On the other hand, in T. infestans the concentration of Nocardia sp. was reduced after infection with B. triatomae, but not by T. rangeli nor T. cruzi. In long-term B. triatomae-infected T. infestans, this reduction and a reduced diuretic activity after feeding synergistically lowered the symbiont concentration in the singly deposited feces/urine drops drastically compared to uninfected controls. These data strongly support the theory of the mechanisms of pathogenicity of T. rangeli and B. triatomae for R. prolixus and T. infestans, respectively, that the primal point of attack is the host-specific symbiont, R. rhodnii and Nocardia sp., respectively.     

RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 microg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75+/-14% and that feeding 1 microg/microL of NP2 dsRNA into second-instar nymphs (approx. 13 microg in total) reduced gene expression by 42+/-10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.     

Isozyme variability and differentiation between Rhodnius prolixus, R.robustus and R.pictipes, vectors of Chagas disease in Venezuela

Enzyme polymorphism in triatomine bugs of the genus Rhodnius (Hemiptera: Reduviidae), vectors of Chagas disease, is analysed using both starch and polyacrylamide gel electrophoresis. Out of forty-five enzymes assayed, the electromorphs of seventeen of them: AO, CA, DIA, ES, ES-A, FH, GPD, G6PD, GPI, MDH, ME, 6PGD, PGM, ACON, ACPH, LAP and SOD, involving twenty-two putative structural loci, were scorable. These gene-enzyme systems were therefore selected for routine characterization of R.prolixus Stal adults from different strains. The first thirteen enzymes, involving sixteen structural loci, were also analysed in first instar nymphs of the three species, R.prolixus, R.robustus Larrousse and R.pictipes Stal. Allelic frequencies were calculated for three R.prolixus strains: three to five loci appeared to be polymorphic. The proportion of polymorphic loci (22%) and the average heterozygosity (0.06) indicated low genetic variability, with significant differences between the strains at individual loci. Rhodnius prolixus and R.robustus were found to have identical isozymic patterns. R.pictipes was genetically well differentiated, with twelve diagnostic loci.     

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.     

Activity and residual effect of two formulations of lambdacyhalothrin sprayed on palm leaves to Rhodnius prolixus

The insecticidal activity and residual effect of two formulations of lambdacyhalothrin were evaluated with Rhodnius prolixus; laboratory and field tests were conducted in the State of Chiapas, Mexico. The results indicate that the lethal concentrations of the active ingredient of SC (LC50 = 2.37 and LC90 = 8.5 mg, a.i./m2) were 4-8 times than those with the insecticide WP applied on R. prolixus bugs in palm leaves, a common building material for thatched roofs. Other investigators in South America recommended applying 30 mg a.i./m2 in porous materials; we obtained that the products WP and SC were 3.5 and 16 times more effective on palm leaves. Regarding the evaluation of the residual effects in field spraying, there was up to 15 months persistence after the application of WP in two doses (8.6 mg a.i./m2 and 3.752 mg a.i./m2) with SC. We consider R. prolixus highly susceptible to the employed pyrethroids; they could be used to control this vector in the state of Chiapas, Mexico.     

Appetence behaviours of the triatomine bug <Emphasis Type="Italic">Rhodnius prolixus</Emphasis> on a servosphere in response to the host metabolites carbon dioxide and ammonia

A combination of 1,000 ppm CO(2) plus 30-40 ppb NH(3) in an air stream induced Rhodnius prolixus nymphs walking on a servosphere to perform a series of appetence behaviours. Shortly after the onset of stimulation the nymphs turned sharply upwind towards the source of the chemostimuli (within 13 +/- 9 s) from mostly downwind and crosswind walks in the air stream alone. The mean vector angles of these upwind tracks were concentrated in a cone 60 degrees either side of due upwind. The upwind walking bugs stopped more frequently but for a shorter duration and walked at a higher speed than before stimulation. During stops in the presence of the chemostimuli the bugs frequently corrected their course angles and extended their forelegs to reach higher with their antennae in the air. In the air stream alone, R. prolixus nymphs frequently sampled the sphere surface with the antennae and cleaned their antennae with the foreleg tarsi. However, the nymphs only briefly tapped the left or right antennal flagellum on the corresponding first leg tarsus and never touched the servosphere surface in the presence of the chemostimuli. After chemostimulus removal from the air stream the bugs continued to respond with the same appetence responses as during stimulation, but walked more tortuously in a crosswind direction in an effort to regain contact with the chemostimuli.     

Blockades of phospholipase A(2) and platelet-activating factor receptors reduce the hemocyte phagocytosis in Rhodnius prolixus: in vitro experiments

The hemocytes phagocytosis in response to microorganisms may play an important role in the cellular immune responses of insects. Here, we have evaluated the effects of the platelet-activating factor (PAF) and eicosanoids in the phagocytosis of hemocyte monolayers of Rhodnius prolixus to the yeast Saccharomyces cerevisiae. Experiments showed that the phagocytosis of yeast cells by Rhodnius hemocytes is very efficient in both controls and cells treated with PAF and arachidonic acid. Phagocytosis of yeast particles is significantly blocked when the specific phopholipase A(2) inhibitor, dexamethasone, is applied on the hemocytes. By contrast, dexamethasone-pretreated hemocyte monolayers exhibit a drastic increase in the quantity of yeast cell-hemocyte internalization when the cells are treated by arachidonic acid. In addition, phagocytosis presents significant reduction in hemocyte monolayers treated with a specific PAF receptor antagonist, WEB 2086. Nevertheless, inhibition of phagocytosis with WEB 2086 is counteracted by the treatment of the hemocyte monolayers with PAF. In conclusion, phagocytosis of yeast cells by hemocytes is related to the activation of PAF receptors and eicosanoid pathways in the bloodsucking bug, R. prolixus.     

The distribution and function of serotonin in the large milkweed bug,Oncopeltus fasciatus. a comparative study with the blood-feeding bug,Rhodnius prolixus

The blood-feeding hemipteran, Rhodnius prolixus, ingests a large blood meal at the end of each larval stage. To accommodate and process this meal, its cuticle undergoes plasticisation, and its gut and Malpighian tubules respectively absorb and secrete a large volume of water and salts for rapid diuresis. Serotonin has been found to be integral to the feeding process in this animal, along with a diuretic peptide(s). The large milkweed bug, Oncopeltus fasciatus, tends to feed in a more continuous and abstemious manner, and therefore may have different physiological requirements than the blood feeder. Unlike R. prolixus, O. fasciatus is lacking serotonin-like immunoreactive dorsal unpaired median neurons in the mesothoracic ganglionic mass, and lacks serotonin-like immunoreactive neurohaemal areas and processes on the abdominal nerves, integument, salivary glands, and anterior junction of the foregut and crop. The salivary glands and crop do, however, respond to serotonin with increased levels of cAMP, while the integument and Malpighian tubules do not. In addition, O. fasciatus Malpighian tubules respond to both O. fasciatus and R. prolixus partially purified CNS extracts, which are likely to contain any native diuretic peptides. Thus, while serotonin and diuretic peptides may be involved in tubule control in R. prolixus, the latter may be of greater importance in O. fasciatus.     

Binding of Lectins to Culture and Vector Forms of Trypanosoma rangeli Tejera, 1920 (Protozoa, Kinetoplastida) and to Structures of the Vector Gut

Culture forms of Trypanosoma rangeli could be agglutinated with Canavalia ensiformis (Con A) lectin and, less effectively with Pisum sativum agglutinin (PEA), at a concentration of 200 μg/ml. Ricinus communis agglutinin I (RCA I) agglutinated trypanosomes only if they were not previously washed with physiological Ringer's solution. Three other lectins did not react with the same parasite forms. Direct or indirect lectin-gold labeling techniques were applied to LR-White embedded thin sections of T. rangeli culture forms and to forms in the gut, hemolymph, and salivary glands of Rhodnius prolixus. Under these conditions, Con A was the only lectin out of 9 that bound to the surface of trypanosomes from culture and from the bug hemolymph. Con A did not react with any midgut or salivary gland forms. The preservation of the biological activity of the lectin-gold complexes that did not bind to the parasite surface was confirmed by reactions with structures of the invertebrate host.     

The effects of aposymbiosis and of an infection with Blastocrithidia triatomae (Trypanosomatidae) on the tracheal system of the reduviid bugs Rhodnius prolixus and Triatoma infestans

We have investigated the effects of the absence of symbionts and of infection with a trypanosome on the tracheal supply to different organs in larvae of the blood-sucking bugs, Rhodnius prolixus and Triatoma infestans. In bugs grown without symbionts there were extensive reductions in the tracheal supply to all the internal organs examined. These bugs also excreted less fluid after blood meals, perhaps because of a reduction in the oxygen supply to the Malpighian tubules. Inclusion of vitamin B in the diet of these affected insects reversed both the adverse effects on diuresis and on the extent of the respiratory supply to the different internal organs. The results of these studies suggest that vitamin B may play a key role in the development and maintenance of an adequate tracheal supply to the tissues in these insects. Infection with the trypanosome Blastocrithidia triatomae also greatly reduced the density of tracheoles supplying the rectum, small intestine and Malpighian tubules in infected T. infestans, but not in R. prolixus. It is possible, therefore, that the parasite exerts at least part of its pathogenic effect by causing a vitamin B deficiency, that in turn affects oxygen supply to the tissues.     

The role of salivary nitrophorins in the ingestion of blood by the triatomine bug Rhodnius prolixus (Reduviidae: Triatominae)

To assist haematophagy, Rhodnius prolixus produces several bioactive molecules in its saliva which it injects into the host skin. The most abundant of these molecules are the nitrophorins (NPs). In this work, we reduced the expression of NP1-4 in the saliva of R. prolixus by RNAi and evaluated the subsequent feeding performance of the bugs using the cibarial pump electromyogram either on the dorsal skin or on the tail vein of the mice. NPs salivary mRNA was reduced by >99% in comparison to controls. Saliva from knockdown nymphs also presented 82% less haemproteins while the total protein was not reduced. Knockdown nymphs feeding on the skin had lower ingestion rates mainly due to the longer cumulative probing time and lower cibarial pump frequency. Another difference was that knockdown insects bit approximately 5 times more. No differences were observed between groups fed on the tail vein. When the feeding sites were compared, nymphs fed on the tail vein had higher effective ingestion rates. These findings endorse the importance of the NPs for the ability of bugs to complete the meal in a short total contact time with a low number of bites, decreasing the perception of the insect by the host.     

Purification and properties of a storage protein from the hemolymph of Rhodnius prolixus

A non-arylphorin hexameric storage protein (SPR) has been isolated from the larval hemolymph of the bug, Rhodnius prolixus. The purified protein contained 0.69% lipid and 1.04% carbohydrate. SPR had a mol. wt of 470,000, with a subunit mol. wt of 78,000. In electron micrographs, SPR molecules appeared very similar to calliphorin. After the metamorphic molt, SPR persisted at lower concentrations in the hemolymph of adult insects, and it disappeared only in fasting bugs.     

Salivary apyrase of Rhodnius prolixus. Kinetics and purification.

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5'-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.     

Binding of lectins to culture and vector forms of Trypanosoma rangeli Tejera, 1920 (Protozoa, Kinetoplastida) and to structures of the vector gut

Culture forms of Trypanosoma rangeli could be agglutinated with Canavalia ensiformis (Con A) lectin and, less effectively with Pisum sativum agglutinin (PEA), at a concentration of 200 micrograms/ml. Ricinus communis agglutinin I (RCA I) agglutinated trypanosomes only if they were not previously washed with physiological Ringer's solution. Three other lectins did not react with the same parasite forms. Direct or indirect lectin-gold labeling techniques were applied to LR-White embedded thin sections of T. rangeli culture forms and to forms in the gut, hemolymph, and salivary glands of Rhodnius prolixus. Under these conditions, Con A was the only lectin out of 9 that bound to the surface of trypanosomes from culture and from the bug hemolymph. Con A did not react with any midgut or salivary gland forms. The preservation of the biological activity of the lectin-gold complexes that did not bind to the parasite surface was confirmed by reactions with structures of the invertebrate host.