Effect of azadirachtin on the development of Trypanosoma cruzi in different species of triatomine insect vectors: long-term and comparative studies.

Studies were carried out on the effects of azadirachtin on the course of Trypanosoma cruzi infection in the gut of different triatomine vector species. In Rhodinus prolixus the development of T. cruzi clone Dm28c decreased in a dose-dependent manner, and the ED50 of this inhibitory effect was 0.25 microgram azadirachtin/ml bloodmeal. Using this insect, we conducted a long-term experiment which showed that azadirachtin (1.0 microgram/ml bloodmeal) completely blocks the development of T. cruzi even 120 days after treatment with the drug, and after four infectant meals. Similarly, the elimination of T. cruzi in feces and urine was also completely blocked over a period of 50 days after infection in insects treated with azadirachtin. Fifth-instar larvae of R. prolixus, Triatoma infestans, and Dipetalogaster maximus, infected with different clone/strains of T. cruzi, displayed drastic inhibition of trypanosome development when treated with azadirachtin. The discussion of these results focuses on the possibility that azadirachtin may act directly on gut physiology and/or indirectly through the neurosecretory system.     

Biological effect of 1-dodecanol in teneral and post-teneral Rhodnius prolixus and Triatoma infestans (Hemiptera: Reduviidae)

Topical application of 1-dodecanol was significantly more toxic against teneral first nymphs (1-3 h old) than post-teneral first nymphs (24 h old). The lethal dose ratios were 711,500 for Rhodnius prolixus and 3613 for Triatoma infestans. No significative difference between LD50 was found when 1-dodecanol was injected in recently hatched adult R. prolixus (1-4 h old) nor in older adults (24 h old). These values were similar to those calculated for deltamethrin (an effective triatomicide), showing that 1-dodecanol had no insecticidal properties when it was applied by injection. Topical application of high dose of 1-dodecanol (1 microg/i) on teneral first nymphs of R. prolixus, produced an interruption of the darkening process of the cuticle, and probably in the development of its physiological properties.     

The biochemical basis of tolerance to malathion in Rhodnius prolixus.

1. LC50 of malathion, fenitrothion and lindane were determined in R. prolixus and T. infestans. R. prolixus was shown to be tolerant to malathion. 2. The penetration rate of (14C)-malathion into R. prolixus and T. infestans was similar. 3. Acetylcholinesterase from R. prolixus heads was 3.3-fold less sensitive to inhibition by malaoxon than the similar enzyme of T. infestans. 4. R. prolixus showed more activity of GSH-S-transferases against DCNB than T. infestans. 5. The in vitro degradation of (14C)-malathion demonstrated that R. prolixus is more active than T. infestans in carboxyester splitting to give alpha and beta monoacids. 6. The synergism of TPP and TOCP on malathion toxicity was higher in R. prolixus than in T. infestans. 7. Esterase activity against alpha and beta naphthyl acetates proved to be much lower in R. prolixus homogenates than in T. infestans homogenates. An inverse result was observed when PTA was the substrate.     

Aggregation behaviour and interspecific responses in Rhodnius prolixus stål

The response to intra- and interspecific faecal assembling signals was tested in Rhodnius prolixus. Papers impregnated with excrement of R. prolixus induced the aggregation of larvae of this species, but also of those of Triatoma infestans. However, faeces belonging to T. infestans were not able to assemble larvae of R. prolixus. On the other hand, there was no response of R. prolixus to putative chemical factors from their cuticle (footprints), in contrast to T. infestans. Results are discussed as related to the ecology of both species.     

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.     

The distribution of agglutinins and lytic activity against Trypanosoma rangeli and erythrocytes in Rhodnius prolixus and Triatoma infestans tissue extracts and haemolymph.

Haemolymph, heads, salivary glands, crops, midguts, hindguts, and Malpighian tubules from Rhodnius prolixus and Triatoma infestans were extracted in phosphate or Tris buffer saline with calcium, and tested for agglutination and lytic activities by microtitration against both vertebrate erythrocytes and cultured epimastigote forms of Trypanosoma rangeli. Haemagglutination activity against rabbit erythrocytes was found in the crop, midgut and hindgut extracts of T. infestans but only in the haemolymph of R. prolixus. Higher titers of parasite agglutinins were found in R. prolixus haemolymph than T. infestans, whilst the converse occurred for the tissue extracts. In addition, the extracts of T. infestans salivary glands, but not those of R. prolixus, showed a trypanolytic activity that was heat-inactivated and was not abolished by pre-incubation with any of the sugars or glycoproteins tested. T. infestans, which is refractory to infection by T. rangeli, thus appears to contain a much wider distribution of agglutinating and trypanolytic factors in its tissues than the more susceptible species, R. prolixus.     

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.     

Inter-specific and developmental differences on the array of antennal chemoreceptors in four species of Triatominae (Hemiptera: reduviidae)

The aim of the work was to investigate the pattern of chemoreceptor sensilla in adults and fifth stage nymphs of Rhodnius prolixus, R. neglectus, Triatoma infestans and T. sordida in order to study differences and similarities between genera and species. Three types of sensilla were analyzed by light microscopy: thin-walled trichoidea, thick-walled trichoidea and basiconica. The number of sensilla of each three types were counted. The length of the antennal segments were also used as a variable for the analysis. The statistical analysis showed that the number of these antennal chemoreceptors had significant differences between species and between adults and nymphs of each species. Discriminant analysis separates incompletely the fifth stage nymphs of the four species and showed similarity between them. Discriminant analysis performed with 12 variables of the antennae, allowed a complete separation of the adults of the four species.     

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.     

Efficacy of ivermectin against the bloodsucking insect, Rhodnius prolixus (Hemiptera, Triatominae)

Ivermectin (0.2 mg/kg body weight) caused a high mortality in nymphs and adults of Rhodnius prolixus following a single meal in mice sub-cutaneously injected with the drug. This effect was more evident in nymphs of 1st-and 2nd-instar than in older nymphs and adults. Third-instar nymphs presented a high mortality when fed on mice treated with ivermectin 24 and 48 hours previously, while mortality was significantly reduced in nymphs fed on mice treated 72 hours before. Surviving 3rd-instar nymphs did not molt. When adult females were fed once on mice treated for 24 hours with ivermectin there was a considerable reduction in egg production. This inhibition was not reversed by a second feeding on normal mice. We concluded that sub-lethal doses of ivermectin caused toxic effects interfering in the neuro-endocrine control of development and reproduction of this bloodsucking insect.     

Effect of sequential cold shocks on survival and molting rate in Triatoma infestans klug

The survival and molting incidence in Triatoma infestans, a vector of Chagas disease, were investigated following sequential shocks at 0 degrees C in fifth instar nymphs under moderate fasting and full nutritional conditions. The shocks were separated by intervals of 8 h and 24 h at 30 degrees C. The results indicated that in terms of insect survival, T. infestans is tolerant to a single cold shock at 0 degrees C even for 12 h, or to sequential cold shocks, regardless of the nutritional state of the specimens. In terms of molting rate, fasting enhanced the tolerance to sequential cold shocks, but did not exceed the tolerance acquired by fully-nourished specimens, except when cold shocks were separated by an 8 h interval at 30 degreesC. The protective action elicited by fasting was assumed to be additive to that induced by a single mild cold shock or sequential cold shocks. The cold-tolerance response of T. infestans may have favoured its survival in areas of South America with low temperatures, even considering that this species is predominantly associated with human habitats.     

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.     

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.     

Defecation index and reproductive success of Triatoma maculata (Hemiptera: Reduviidae) under laboratory conditions

The reproductive and defecating behavior of Triatoma maculata (Erichson 1848) was studied on animals from an university culture in Venezuela. This species does not reach the importance of Rhodnius prolixus Stal 1859 as Chagas disease vector in Venezuela. This study addressed the role of defecating frequency, an index of how dangerous the animals are for the human population, and its relationship with why T. maculata is a less important vector than R. prolixus. Human blood was fed to the insects through an artificial feeding device. The 2nd and 3rd instar nymphs defecated more frequently (Id= 0.6, n=40), and our Vth instar nymphs did not complete sexual differentiation. Fertility was 55% (n=865) and fecundity 8 eggs/female/week (n=26). Egg incubation lasted 22 days (n=477). Female longevity was 51 days (n=26). Intermould time grew progressively from 35 days for 1st to 40 days for 4th instar nymphs (n=40). Mould percentage varied from 0% for Vth to 63% for 3rd instar nymphs. Mortality varied from 8% for 3rd to 100% for Vth instar nymphs. These results support evidences explaining the lesser vectorial capacity and low density of T. maculata in human domiciles, including reduced reproduction and defecation when the animal feeds on human blood.     

Effects of fluctuating moisture and temperature regimes on the infection potential of Beauveria bassiana for Rhodnius prolixus

The effect of both moisture and temperature on the infective potential of Beauveria bassiana to the Chagas' disease vector, Rhodnius prolixus, was studied under fluctuating regimes. At constant 25 degrees C, contaminated first-instar nymphs exposed to increasing daily periods of initial exposure to 97% RH, followed by transfer to reduced humidity (43, 53, 75, and 86% RH), showed a significant reduction in mortality when the 97% RH exposure time declined from 12 to 8 h per day. The duration of disease incubation depended on the daily 97% RH exposure time. Under fluctuating regimes of both humidity (97% RH versus 75% RH) and temperature (15/28, 20/25, 25/28, and 25/35 degrees C), first-instar mortality was affected by weather conditions, daily 97% RH exposure time (8, 12, and 16 h per day), and number of temperature and humidity fluctuations before transferring tested insects to constant unfavorable conditions. In most cases, at 12/12 h alternating cycles, high and rapid mortality required five cycles. Under these fluctuating regimes, fungus-induced mortality and mortality time were similarly affected in third- and fifth-instar nymphs by the daily 97% RH exposure time. Despite a lower susceptibility of older larval stages, mortality rates in insects exposed for at least 12 h per day at 97% RH remained very high except at 15 degrees C. Moisture and temperature regimes at 12/12 h cycling significantly affected the dose-mortality response in first-instar nymphs. The most favorable conditions consisted of 97%-20 degrees C combined with either 75%-25 degrees C or 43%-25 degrees C. Under less favorable alternating conditions (lower and higher temperatures) the amounts of inoculum required for killing 50% of first-instar nymphs were 10 or 20 times higher. From a vector control standpoint, daily high humidity appears to be the most crucial climatic constraint. B. bassiana has the potential to control R. prolixus populations with applications made during the rainy seasons when humidity is high.     

Role of superoxide and reactive nitrogen intermediates in Rhodnius prolixus (Reduviidae)/Trypanosoma rangeli interactions.

This study compares aspects of the superoxide, nitric oxide and prophenoloxidase pathways in Rhodnius prolixus hemolymph, measured in parallel, in response to Trypanosoma rangeli inoculation. Responses to two strains of T. rangeli, and two developmental forms, were studied, and the results obtained were correlated with the ability of the parasites to survive, multiply, and complete their life cycles in the hemolymph of the host. T. rangeli H14 strain parasites, which fail to complete their life cycle in Rhodnius by invading the salivary glands, stimulated high levels of superoxide and prophenoloxidase activity, which peaked 24 h after inoculation. Simultaneously, the concentration of hemolymph nitrites and nitrates increased, indicative of nitric oxide activity, but parasite numbers remained low. T. rangeli Choachi strain parasite inoculation also stimulated superoxide and prophenoloxidase activity, which, though significantly lower than the equivalent responses to the H14 strain, also peaked at 24 h. However, nitrate and nitrite levels in Choachi strain-inoculated hemolymph remained low, and this parasite strain multiplied rapidly, especially following peak superoxide activity, and eventually invaded the salivary glands for transmission to a vertebrate host. In both strains, short form epimastigotes stimulated greater superoxide and prophenoloxidase responses than long form epimastigotes. Injection of the NADPH oxidase inhibitor N-ethylmaleimide or the inducible nitric oxide synthase inhibitor S-methyl isothiourea sulfate caused significantly higher insect mortalities in groups of R. prolixus inoculated with either parasite strain compared with those of uninfected control insects. This indicates that both NADPH oxidase and nitric oxide synthase activity may be involved in the immune response of R. prolixus to infection by T. rangeli. Finally, Western blotting of R. prolixus hemocyte lysates revealed the presence of a protein immunologically related to the human NADPH oxidase complex, the initiator enzyme of the respiratory burst.     

Changes in salivary nitrophorin profile during the life cycle of the blood-sucking bug Rhodnius prolixus

The insect Rhodnius prolixus is a hematophagous hemipteran that has five nymphal instars. Fifth instar nymphs contain, in their salivary glands, four nitrophorins which have already been described in the literature (NP1, NP2, NP3 and NP4). Two new hemeproteins were isolated and partially characterized from first instar nymphs. NP2, that shows an anticoagulant activity, was also identified, but NP1, NP3 and NP4 were not found. As these new hemeproteins have amino-terminal sequences clearly homologous to already described nitrophorins and were capable of binding nitric oxide, they were named nitrophorins 5 and 6, although they showed an unusual Soret band at 412 nm. In each subsequent nymphal stage, a new nitrophorin emerges. In the second instar, NP4 comes into view, in the third instar NP1 appears, and NP3 is only found in fifth instar nymphs and adults, showing that the nitrophorin profile of R. prolixus saliva is stage-specific.     

Residual effect of a micro-encapsulated formulation of organophosphates and piriproxifen on the mortality of deltamethrin resistant Triatoma infestans populations in rural houses of the Bolivian Chaco region

The Bolivian Chaco is part of the endemic region of Chagas disease and an area where pyrethroid resistant Triatoma infestans (Hemiptera: Reduviidae) populations has been reported. The World Health Organization identified these resistant populations as an important focus for research. The objective of this study was to evaluate the residual effect of a micro-encapsulated formulation containing organophosphate active ingredients and a juvenile hormone analogue (Inesfly 5A IGR) on the mortality of T. infestans. Studies took place in rural houses of the Bolivian Chaco that were treated up to 34 months before and evaluated the susceptibility to pyrethroids of the offspring of field collected insects. Thirty houses were randomly selected within three communities to carry out wall bio-assays with T. infestans nymphs. Mortality was recorded 24, 48 and 72 h after wall contact. Eggs laid by females collected in the area were used to obtain first-instar nymphs and carry out pyrethroid susceptibility tests. The wall bio-assays showed that the micro-encapsulated insecticide eliminates T. infestans populations and produces detectable mortality of insects exposed to walls treated 34 months prior to the tests. The discriminant dose of deltamethrin (0.01 mg/mL) showed 65% nymph survival, whereas at the highest tested dose (1.0 mg/mL) 14% of the nymphs survived. These results show that Inesfly 5A IGR is an appropriate tool for the elimination of intradomestic and peridomestic populations of T. infestans resistant to pyrethroids.     

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.     

Changes in the rate of CO2 release following feeding in the insect Rhodnius prolixus

We describe for the first time changes in the rate of CO2 release (as a surrogate of metabolic rate) in the terminal larval stage of the insect Rhodnius prolixus following a blood meal and during the molt leading to the adult stage. These data are presented on a whole-animal basis as well as per gram wet and dry weight. We have also used techniques that allow us to describe the rate of release per gram of actual body tissue (i.e., removing the weight of the remaining bloodmeal in the gut and the metabolically inactive portion of the cuticle). While the metabolic rate of the whole animal rises approximately 10-fold in 15 d following feeding, the rate per gram of dry body mass rises only twofold. We use these data to provide insights into the relative contributions of tissue growth and increases in metabolic intensity to the massive increases in metabolic rate observed in these insects following feeding. Our analyses indicate that the majority of nutrient uptake occurs in the first 4 d following feeding. It is well known in this species that day 4 following feeding is the end of a critical period for the insect in determining whether it will proceed to the next molt. Our results indicate that the insects may be able to make this decision based on nutrients already transported into the body. We examined the "down regulation" of metabolism observed in the latter stages of the molt cycle in this insect. We express these changes on both a per animal and per gram basis and demonstrate that this down regulation extends even into the adult stage before feeding. Using a comparison of the allometric relationships of metabolic rate to mass in insects and ticks, we demonstrate that unfed R. prolixus show a marked decrease in metabolic rate compared to other insects, while fed Rhodnius are similar in metabolic rate to other insects. Rhodnius has a markedly higher metabolic rate (as do all insects) than that found in ticks.