Ultrastructural and enzymatic alterations in the ovary of Rhodnius prolixus infected with Trypanosoma rangeli

Objective: To investigate the morphological structure of ovarian follicular cells and biochemical parameters of both ovaries and fat bodies (sites of vitellogenesis) from Rhodnius (R.) prolixus infected with Trypanosoma (T.) rangeli. Methods: Adult virgin females of R. prolixus were fed upon a membrane apparatus containing heat-inactivated citrated rabbit blood and a suspension of T. rangeli epimastigotes (Macias strain). Females from the control group and all the males received parasitefree blood. Transmission electron microscopy was used to reveal the morphological aspects of ovarian follicle cells in both control and parasite-infected groups. Protein profile, proteolytic activities and Western blotting analyses were performed in either ovary or fat body samples of control and parasite-infected groups. Results: According to the ultrastructural data, T. rangeli infection elicited a degeneration process in the ovarian follicular cells of R. prolixus. Proteolytic assays indicated a reduction in the activity of aspartic peptidases in the ovary and fat body from parasite-infected group, while a significant increase in the cysteine peptidase activity was measured in both insect organs. Additionally, immunoblotting revealed that vitellogenin was overexpressed in the ovary of parasite-infected insects. Conclusions: T. rangeli infection seems to elicit an early programmed cell death in the ovarian follicle cells as well as induces the modulation on the activities of different peptidase classes in either ovaries or fat bodies and the overexpression of the vitellogenin in the ovary of R. prolixus.

Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase

Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods: We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results: Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation. Conclusion: These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.(AU)

Lethal and sublethal effects of essential oil of Lippia sidoides (Verbenaceae) and monoterpenes on Chagas´ disease vector Rhodnius prolixus

The aim of this study was to identify the composition of the essential oil from leaves of Lippia sidoides (EOLS), a typical shrub commonly found in the dry northeast of Brazil, popularly known as “alecrim-pimenta”. Additionally, we investigated the nymphicidal, ovicidal, phagoinhibitory and excretion effects of EOLS, its major constituent thymol and its isomer carvacrol, on fourth instar nymphs and eggs of Rhodnius prolixus, the Chagas’ disease vector. The nymphicidal and ovicidal activity of thymol, carvacrol, and EOLS was assessed by tests using impregnated Petri dishes. The lethal concentration values (LC50) for EOLS, carvacrol, and thymol were 54.48, 32.98, and 9.38 mg/cm2, respectively. The ovicidal test showed that both carvacrol and thymol (50 mg/cm2) inhibited hatching (50% and 23.3%, respectively), while treatments with 10 mg/cm2 or 50 mg/cm2 EOLS did not affect the hatching rate at all (80% and 90%, respectively). We observed an anti-feeding effect in insects fed with blood containing natural products at the higher concentrations (100 µg/mL). Finally, excretion rate was affected by EOLS and carvacrol, but not by thymol. These findings offer novel insights into basic physiological processes that make the tested natural compounds interesting candidates for new types of insecticides.

Behavioral fever response in Rhodnius prolixus (Reduviidae: Triatominae) to intracoelomic inoculation of Trypanosoma cruzi

Abstract: INTRODUCTION: Behavioral fever is a response to infections with microorganisms observed in some poikilothermic animals. Rhodnius prolixus is involved in the transmission of two parasites: Trypanosoma cruzi (pathogenic for humans and transmitted in feces) and Trypanosoma rangeli (non-pathogenic for humans, pathogenic for Rhodnius and transmitted by the bite of an infected individual). Only T. rangeli is found in the hemolymph of Rhodnius as it travels to the salivary glands. METHODS: To study vector-parasite interactions, we evaluated possible behavioral fever responses of R. prolixus to intracoelomic inoculation with T. cruzi or T. rangeli. Temperature preferences of fifth-instar nymphs of R. prolixus were evaluated after inoculation with T. rangeli KP1(+), KP1(-), T. cruzi I, or the Trypanosome culture medium. Four different fixed temperatures (25, 30, 35, and 40°C) in two simultaneous experiments (enclosed and free-moving insects) were evaluated. Free-moving insects were marked daily according to their temperature preferences on each of the 15 days after inoculation. Numbers of insects in each temperature shelter and daily mortality were compared with those enclosed shelters of different temperatures. RESULTS: Rhodnius prolixus inoculated with both strains of T. rangeli and with the trypanosome culture medium showed preferences for the lowest temperatures (25°C). However, R. prolixus inoculated with T. cruzi I showed significant preferences for temperatures around 35°C. CONCLUSIONS: This is the first known investigation to demonstrate a behavioral fever response in R. prolixus injected intracoelomically with T. cruzi I.

Estrategia para determinar la línea base en áreas de interrupción vectorial de la enfermedad de Chagas / Strategy for determining a baseline in areas of vector interruption for Chagas disease

RESUMEN Objetivos Presentar una estrategia para determinar la línea de base en áreas endémicas en el proceso de interrupción vectorial de la enfermedad de Chagas (ECh). Métodos Con un cuestionario socioambiental y una encuesta entomológica, se evaluaron las condiciones físicas de los domicilios, el conocimiento de los habitantes sobre la ECh, los indicadores entomológicos de triatominos y la relación estadística entre estas variables. Resultados Existe colonización e infección natural con Trypanosoma cruzi en Rhodnius prolixus, el principal vector de la ECh en Colombia. La colonización estuvo relacionada con casas de techos de hojas de palma y paredes de adobe o bahareque. Se encontró el vector Panstrongylus geniculatus con hábitos de colonización. Casi 50% de la población encuestada asociaba el término ECh con una enfermedad humana y 37%, con los triatominos. Conclusiones R. prolixus puede considerarse el principal vector de T. cruzi en ambientes domésticos y el proceso de interrupción vectorial es factible dentro del municipio priorizado. Se necesitan nuevos estudios que comprueben la existencia de poblaciones silvestres de R. prolixus que puedan afectar las etapas futuras del proceso y demostrar si hay una participación de P. geniculatus en la dinámica de transmisión de T. cruzi. Los anteriores escenarios pueden ser viabilizados con la inclusión de los habitantes en todo el proceso, ya que han sido altamente sensibles en la detección de vectores dentro de sus casas. El diseño del estudio aquí presentado puede ser adaptado en otras áreas endémicas de la Región de las Américas.

ABSTRACT Objectives Present a strategy to determine the baseline in endemic areas in the process of vector interruption for Chagas disease (CHD). Methods A social and environmental questionnaire and an entomological survey evaluated the physical conditions of dwellings, the inhabitants’ knowledge of CHD, the entomological triatomine indicators and the statistical relationship among these variables. Results Colonization and natural infection with Trypanosoma cruzi exist in Rhodnius prolixus, the principal vector of CHD in Colombia. Colonization was related to palm-thatched houses constructed with adobe or wattle and daub. The Panstrongylus geniculatus vector was found to be colonizing. Almost 50% of the surveyed population associated the term CHD with human disease and 37%, with triatomines. Conclusions R. prolixus can be considered to be the principal vector of T. cruzi in domestic environments and the process of interruption is feasible within the prioritized municipality. New studies are needed to verify the existence of wild populations of R. prolixus that could affect future stages of the process and demonstrate whether P. geniculatus is a factor in the transmission of T. cruzi. These scenarios can be made viable by including the inhabitants throughout the process, since they have been highly sensitive in vector detection inside their houses. The study design presented here can be adapted to other endemic areas of the Region of the Americas.

New scenarios of Trypanosoma cruzi transmission in the Orinoco region of Colombia

Rhodnius prolixus, a blood-sucking triatomine with domiciliary anthropophilic habits, is the main vector of Chagas disease. The current paradigm of Trypanosoma cruzi transmission in Columbia includes a sylvatic and domiciliary cycle co-existing with domestic and sylvatic populations of reservoirs. The aim of this study is to evaluate the population densities and relative abundance of triatomines and mammals that may be involved in the sylvatic cycle of Chagas disease to clarify the epidemiological scenario in an endemic area in the province of Casanare. Insect vectors on Attalea butyracea palms were captured using both manual searches and bait traps. The capture of mammals was performed using Sherman and Tomahawk traps. We report an infestation index of 88.5% in 148 palms and an index of T. cruzi natural infection of 60.2% in 269 dissected insects and 11.9% in 160 captured mammals. High population densities of triatomines were observed in the sylvatic environment and there was a high relative abundance of reservoirs in the area, suggesting a stable enzootic cycle. We found no evidence of insect domiciliation. Taken together, these observations suggest that eco-epidemiological factors shape the transmission dynamics of T. cruzi, creating diverse scenarios of disease transmission.

Comparison of hemocytes of V-instar nymphs of Rhodniusprolixus (Stål) and Rhodnius robustus (Larousse 1927), before and after molting / Comparación de los hemocitos de las ninfas de V instar de Rhodnius prolixus (Stål) and Rhodnius robustus, antes y después de la muda (Larousse 1927)

Background. The immune response of insects involves humoral factors and cellular elements known as hemocytes. There are different reports that explore the response of hemocytes to infections, but the effect that molting has on this response has not been explored so far. We hypothesized that there would be a change in the percentage of hemocytes as a response to the molting process. Objective. The aim of this work was to compare the hemogram (CBC), the formula, and the differential count of hemocytes in IV instar before molting and in V instar nymphs 24 hours after molting in two species: Rhodnius prolixus and Rhodnius robustus. Materials and methods. We assayed different staining methodologies including Giemsa, Alcian Blue pH 2.0, Alcian Blue pH 2.6, Gomori substrate, PAS (Schiff), Sudan Black and Papanicolau with positive controls for each one. In the Gomori staining, we observed lysosomes in the granulocytes and plasmatocytes, but the differentiation was better detected using Giemsa staining. Results. There were no statistically significant differences between the two species studied in plasmatocytes (p=0,053) or even in granulocytes (p=0,5). However, differences were significant in the prohemocytes (p=0,001) during the molting process in both Rhodnius prolixus and Rhodnius robustus. Conclusions. Significant differences in prohemocytes between nymphs of IV and V instar were detected. No significant differences in the amount of cells were observed between the two species and the two stages. These findings may be explained due to their role as precursor cell of prohemocytes.

Antecedentes. La respuesta inmune de los insectos involucra factores humorales y elementos celulares llamados hemocitos. Existen varios trabajos explorando la respuesta de los hemocitos frente a infecciones, pero no como respuesta al proceso de muda. Objetivo. Comparar el hemograma: la fórmula y el recuento diferencial de hemocitos de Rhodnius prolixus y Rhodnius robustus en ninfas de IV estadio antes de la muda y ninfas de V estadio 24 horas después de la muda. Materiales y métodos. Se ensayaron las coloraciones Giemsa, Alcian Blue pH 2.0, Alcian Blue pH 2.6, sustrato Gomori, PAS (Schiff), Sudán Negro, Papanicolau, con controles positivos para cada una. Con la coloración de Gomori se observaron lisosomas en los granulocitos y en plasmatocitos, pero la diferenciación se logró mejor con la coloración de Giemsa. Resultados. Los plasmatocitos fueron más abundantes en las ninfas de IV estadio de Rhodnius robustus y en las de V estadio de las dos especies, pero en las ninfas de V estadio de R. prolixus, el recuento de prohemocitos fue mayor; para los oenocitoides, esferulocitos y adipohemocitos el recuento no alcanzó al 1 %. En las dos especies no se encontraron diferencias estadísticamente significativas en plasmatocitos (p=0,05319), ni en granulocitos (p=0,5), entre las ninfas de IV y V estadio, pero fue significativa en prohemocitos (p=0,001). Conclusiones. Se detectaron diferencias significativas en prohemocitos entre las ninfas de IV y V estadio. No hubo un aumento significativo en el conteo de células en los diferentes estadios.

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.

Impact of a community-based bug-hunting campaign on Chagas disease control: a case study in the department of Jalapa, Guatemala

Chagas disease control requires an innovative approach to strengthen community participation in vector surveillance. This paper presents a case study of a community-based bug-hunting campaign in Guatemala. The campaign was implemented in 2007 in the following three stages: (i) a four week preparation stage to promote bug-hunting, (ii) a one week bug-hunting stage to capture and collect bugs and (iii) a 10 week follow-up stage to analyse the bugs and spray insecticide. A total of 2,845 bugs were reported, of which 7% were Triatominae vectors, such as Rhodnius prolixus and Triatoma dimidiata. The bug-hunting campaign detected a five-six-fold higher amount of vectors in one week than traditional community-based surveillance detects in one year. The bug-hunting campaign effectively detected vectors during a short period, provided information to update the vector infestation map and increased community and political awareness regarding Chagas disease. This approach could be recommended as an effective and feasible strategy to strengthen vector surveillance on a larger scale.

Elimination of Rhodnius prolixus in El Salvador, Central America

Epidemiological studies performed in El Salvador between 1955-1972 demonstrated a high frequency of two triatomine vectors (Triatoma dimidiata and Rhodnius prolixus). However, the frequency of R. prolixus decreased by 1976 and this species has not been found since 1995. The main factors influencing the elimination of R. prolixus in El Salvador are discussed in this paper.

Vector control intervention towards interruption of transmission of Chagas disease by Rhodnius prolixus, main vector in Guatemala

In Guatemala, the Ministry of Health (MoH) began a vector control project with Japanese cooperation in 2000 to reduce the risk of Chagas disease infection. Rhodnius prolixus is one of the principal vectors and is targeted for elimination. The control method consisted of extensive residual insecticide spraying campaigns, followed by community-based surveillance with selective respraying. Interventions in nine endemic departments identified 317 villages with R. prolixus of 4,417 villages surveyed. Two cycles of residual insecticide spraying covered over 98% of the houses in the identified villages. Fourteen villages reinfestated were all resprayed. Between 2000-2003 and 2008, the number of infested villages decreased from 317 to two and the house infestation rate reduced from 0.86% to 0.0036%. Seroprevalence rates in 2004-2005, when compared with an earlier study in 1998, showed a significant decline from 5.3% to 1.3% among schoolchildren in endemic areas. The total operational cost was US$ 921,815, where the cost ratio between preparatory, attack and surveillance phases was approximately 2:12:1. In 2008, Guatemala was certified for interruption of Chagas disease transmission by R. prolixus. What facilitated the process was existing knowledge in vector control and notable commitment by the MoH, as well as political, managerial and technical support by external stakeholders.

Epimastigogénesis de Trypanosoma cruzi: método para estudiar in vivo la transformación de tripomastigota en epimastigota / Trypanosoma cruzi epimastigogenesis: a method for studying Trypomastigote to epimastigote transformation in vivo

La epimastigogénesis de Trypanosoma cruzi ocurre naturalmente en el intestino del hospedador invertebrado. Se desconoce si los cambios morfológicos que ocurren durante la transformación de los tripomastigotas sanguíneos en epimastigotas son idénticos para diferentes aislados de T. cruzi. Aquí mostramos un método útil para estudiar los eventos que ocurren durante la epimastigogénesis, comparando parásitos de diferentes procedencias epidemiológicas. Se alimentaron artificialmente ninfas de V estadio de Rhodnius prolixus con una solución ad hoc conteniendo tripomastigotas tipo-sanguíneo y siguiendo los cambios morfológicos por 8 días. Los contenidos del intestino anterior de las ninfas se obtuvieron decapitando y comprimiendo el abdomen lo que permitió obtener rápidamente casi 100% del inóculo con cargas entre 1,9 y 8,9 × 106 tripomastigotas/ninfa. El número de parásitos por ninfa y los cambios morfológicos se determinaron por Microscopia de Contraste de Fases y coloración con Giemsa. Las ninfas ingirieron 7,4 veces su peso de solución infectante, con volúmenes entre 101 y 357 µL (229 ± 66 mg), 50% de esa ingesta se eliminó como orina durante las primeras 24 h. Los tripomastigotas se transformaron en formas redondeadas antes de evolucionar a epimastigotas, siguiendo cinéticas diferentes según el aislado. Proponemos esta metodología para estudiar rápida y cuantitativamente los eventos tempranos de la epimastigogénesis de T. cruzi in vivo.

Trypanosoma cruzi epimastigogenesis naturally occurs in the intestine of the invertebrate host. It is not known whether the morphological changes that occur during transformation of bloodstream trypomastigotes to epimastigotes are identical for different T. cruzi isolates. This research shows a useful method for studying the events that occur during epimastigogenesis, comparing parasites from epidemiological sources. Rhodnius prolixus V stage nymphs were fed artificially with an ad hoc solution containing blood-like trypomastigotes and the morphological changes were examined during eight days. Anterior intestinal contents were removed by decapitation and squeezing the abdomen of the nymphs, which permitted obtaining quickly almost 100% of the inoculate with loads between 1.9 and 8.9 × 106 trypomastigotes/nymph. The number of parasites per nymph and morphological changes were determined using phase microscopy with Giemsa staining. The nymphs ingested 7.4 times their weight of the infecting solution with volumes between 101 e 357 µL (229 ± 66 mg); 50% of the ingest was eliminated as urine during the first 24 hours. The trypomastigotes transformed to rounded forms before evolving into epimastigotes following different kinetics according to the isolate. This method is proposed for rapid, quantitative study of the early events of epimastigogenesis for T. cruzi in vivo.

Atuação das proteínas que ligam à heparina no ciclo biológico do trypanosoma cruzi / Actions of proteins that bind to heparin in the biological cycle of Trypanosoma cruzi

A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, é uma doença tropical negligenciada que representa um grave problema de saúde pública. Assim, compreender a biologia da interação T. cruzi-hospedeiros constitui um grande desafio, uma vez que o ciclo de vida deste parasito exige um repertório de adaptações para garantir sua dispersão em hospedeiros vertebrados e invertebrados. O presente estudo demonstra o potencial das proteínas com propriedade de ligação à heparina (PLHs) em atuar no ciclo biológico do T. cruzi. Durante este trabalho foi oportuno isolar uma fração de proteínas hidrofóbicas com propriedade de ligação à heparina, com massas moleculares entre 70 kDa e 59 kDa em formas epimastigotas e tripomastigotas de T. cruzi por cromatografia de afinidade à heparina. A presença destas proteínas na superfície celular destes parasitos foi confirmada por ressonância plasmônica de superfície. Tais ensaios também foram decisivos na determinação da especificidade e estabilidade da ligação das PLHs a heparina, heparam sulfato (HS) e condroitim sulfato (CS). Os ensaios de competição realizados indicaram que a interação entre PLHs e GAGs pode influenciar a adesão dos epimastigotas à superfície de células epiteliais do trato intestinal de Rhodnius prolixus...

O envolvimento de GAGs na invasão de amastigotas em cardiomiócitos, célula alvo da infecção pelo T. cruzi, também foi demonstrado através de ensaios de competição com 20 g/ml de GAGs solúveis, incluindo heparina, HS, CS, dermatam sulfato (DS) e queratam sulfato (KS). Uma drástica redução no nível de infecção foi evidenciada apenas com heparina e HS, atingindo 82 por cento e 65 por cento de redução da invasão, respectivamente. Ensaios com células deficientes em GAGs (CHO-745) corroboraram o importante papel destes componentes de matriz extracelular no processo de reconhecimento e invasão de amastigotas. Na continuidade deste estudo, avançamos na caracterização bioquímica de PLHs, na determinação da expressão e distribuição espacial destas proteínas em tripomastigotas. As análises por citometria de fluxo revelaram que PLHs são abundantes na superfície de tripomastigotas, clone Dm28c, e a detecção destas proteínas por imunofluorescência indireta revelou uma localização predominante na membrana flagelar do parasito. Com os ensaios de zimografia realizados neste trabalho, revelamos que as PLHs de tripomastigotas tem atividade de protease sobre gelatina em uma ampla faixa de pH (5,5 - 8,0). A sensibilidade destas enzimas a presença de inibidores de serino protease indicam que..

Behavioural responses to human skin extracts and antennal phenotypes of sylvatic first filial generation and long rearing laboratory colony Rhodnius prolixus

Chagas disease is a major public health issue and is mainly spread by Triatominae insects (Hemiptera: Reduviidae). Rhodnius prolixus is the main vector species in Northern South America. Host-seeking behaviour in R. prolixus is mediated by different compounds that are produced by and emanate from the host or microbiota on the host's skin. We tested the behavioural responses of sylvatic first filial generation (F1) and colony insects to extracts of human skin with a dual choice olfactometer. In addition, we compared the antennal phenotypes in both populations. No statistical differences were found between the two populations at the behavioural level. Both showed a preference for face and feet extracts and this effect was abolished for face extracts after treatment with an antibacterial gel. The observation of the antennal phenotype showed that there were differences between both groups in the total length, total surface area and number and density of bristles. However, the number and density of chemoreceptive sensilla (basiconic and thin and thick-walled trichoids) and the total density of sensilla did not show statistically significant differences. These results demonstrate that colony insects, which have only been fed with living hens for the last 30 years, are attracted by human skin extracts in a similar way as F1 sylvatic insects.

Commercial coconut palm as an ecotope of Chagas disease vectors in north-eastern Venezuela.

Background & objective: There are few reports of Coccus nucifera (Palmae) infestation by triatomines (Hemiptera, Reduviidae, Triatominae), vectors of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae), the causal agent of American Trypanosomiasis. The aim of this study was to determine if this palm is an appropriate ecotope for Rhodnius prolixus and Triatoma maculata, the main vectors in Venezuela. Methods: Dry and green leaves, humid debris, interfoliaceus meshes and bracts from C. nucifera from north-eastern Venezuela were examined for the presence of triatomines. Samples of the intestinal content of vectors, macerated in isotonic saline solution and haemolymph were examined microscopically for the presence of Trypanosoma spp. The parasites were isolated and characterized using biological parameters and PCR. Triatomine blood meal sources were determined using ELISA. Results: A total of 14 palms were examined in which viable eggs of both species of vectors were found in 13 palms (92.85%). A total of 242 R. prolixus and 144 T. maculata adults were collected, of which 98% of R. prolixus and 70% of T. maculata individuals were infected by T. cruzi (TcI genotype) and 13% of R. prolixus individuals showed a mixed infection with T. rangeli, the other American trypanosome. ELISA testing for possible triatomine blood-meal sources revealed that these vectors are essentially eurytrophic and zoophilic, although R. prolixus also eventually used human blood as a nourishment source. Interpretation & conclusion: The results obtained suggest that C. nucifera is an appropriate vegetal niche for these triatomine species in Venezuela. The presence of this commercial palm may represent a useful environmental bioindicator of risk for Chagas disease.

Primary culture of Rhodnius prolixus (Hemiptera: Reduviidae) salivary gland cells

In the present paper, we developed a primary culture of Rhodnius prolixus salivary gland and main salivary canal cells. Cells remained viable in culture for 30 days. Three types of cells were indentified in the salivary gland cultures, with binuclear cells being the most abundant. The supernatants of salivary cultures contained mainly 16-24 kDa proteins and presented anticoagulant and apyrase activities. Secretion vesicles were observed budding from the cellular monolayer of the main salivary canal cells. These results indicate that R. prolixus salivary proteins may be produced in vitro and suggest that the main salivary canal may have a possible secretory role.

Laboratory evaluation of the effects of triflumuron on the development of Rhodnius prolixus nymph

Studies were carried out to evaluate the efficacy of the growth regulator, triflumuron (TFM) (Starycide® sc 480 Bayer), for disrupting the development of Rhodnius prolixus fifth-instar nymph by oral, topical or continuous treatment. All treatments were able to induce high levels of mortality, delay development and molt inhibition. Oral treatment induced molt inhibition in all insects that survived at doses of 0.25, 0.50 and 5.0 mg/mL of a blood meal. The highest levels of both mortality in 24 h and molt inhibition were always observed after topical treatment. The lowest doses needed to obtain considerable biological effects were always observed after continuous treatment. In this way, the highest levels of mortality within 30 days were detected after continuous treatment, which also induced an extended inter-molting period, a lower number of over-aged nymphs and the highest level of molting in nymphs that survived. Moreover, the effects of TFM on insects were often displayed in a dose response manner. These results indicate that TFM acts as a potent growth inhibitor of R. prolixus nymphs and has the potential to be used in integrated vector control programs against hematophagous triatomine species.

Preliminary characterization of a Rhodnius prolixus hemolymph trypanolytic protein, this being a determinant of Trypanosoma rangeli KP1(+) and KP1(-) subpopulations' vectorial ability

Rhodnius prolixus is the main Trypanosoma rangeli vector in several Latin-American countries and is susceptible to infection with KP1(+) strains; however, it presents an invasion-resistant response to KP1(-) strains. The present work has identified a trypanolytic protein against T. rangeli KP1(-) in the R. prolixus hemolymph which was fractioned with ammonium sulfate (following dialysis). The results revealed a protein component which did not depend on divalent cations for its biological function whilst keeping its trypanolytic activity at temperatures ranging from -20ºC to 37ºC, at 7.0 to 10.5 pH. The protein was partially purified by gel filtration chromatography and ionic exchange chromatography. The major component presented a molecular weight of around 79 kDa and an isoelectric point between 4.9 and 6.3 and may be directly related to hemolymph trypanolytic activity against T. rangeli KP1(-) populations.

Effects of the essential oil obtained from Pilocarpus spicatus Saint-Hilaire (Rutaceae) on the development of Rhodnius prolixus nymphae

Bioassays against fifth-instar nymphae of Rhodnius prolixus were conducted with essential oil of Pilocarpus spicatus extracted by hydrodistillation. The main results may be summarized as follows: (i) high levels of toxicity and paralysis together with discrete moulting inhibition were caused by topical application of either 0.5 µL or 1.0 µL per insect of the crude essential oil; (ii) partial fagoinhibition, high moulting inhibition, prolonged intermoulting period and high number of paralyzed insects, but no toxicity were observed after oral treatment using either 5 µL or 10 µL of Pilocarpus spilcatus essential oil per mL of ingested blood meal. The importance of these results in relation to the relevant biological events in R. prolixus is herein discussed.

Bioensaios contra ninfas de 5º estádio de Rhodnius prolixus foram conduzidos utilizando-se óleo essencial de Pilocarpus spicatus extraído por hidrodestilação. Os principais resultados podem ser resumidos como se segue: (i) altos níveis de toxicidade e paralisia associados à discreta inibição da muda foram induzidos pela aplicação tópica de 0,5 µL ou 1,0 µL do óleo essencial por inseto; (ii) fagoinibição parcial, altos níveis de inibição da muda, período intermuda prolongado e alto número de insetos paralisados mas ausência de toxicidade foram observados após tratamento oral com 5,0 µL ou 10 µL de óleo essencial de P. spicatus por mL de sangue ingerido. A importância destes resultados em relação a eventos biológicos relevantes em R. prolixus é aqui discutida.

Effects of azadirachtin in Rhodnius prolixus: data and hypotheses

The effects of azadirachtin A, a tetranortriterpenoid from the neem tree Azadirachta indica J., on both development and interaction between Trypanosoma cruzi, the causative agent of Chagas' disease, and its vector Rhodnius prolixus were studied. Given through a blood meal, a dose-rsponse relationship of azadirachtin was established using antifeedant effect and ecdysis inhibition as effective parameters. A singlo dose of azadirachtin A was able to block the onset of mitosis in the epidermis and ecdysteroid titers in the hemnolymph, determined by radioimmuneassay, were too low for an induction of ecadysis. The survival of T. cruzi was also studied in R. prolixus treated with the drug. If the trypomastigotes were fed in presence of azadirachtin A the number of parasites drastically decreased. If the drug was applied after infection of the bug with T. cruzi, the parasite was still abolished from the gut. If the insect was pretreated with azadirachtin A before infection the same observation was obtained. A single dose of azadirachtin A was enough for a permanent resistance of the insect host against its reinfection with T. cruzi and for blocking the ecdysis for a long time. The effects of azadirachtin A on the hormonal balance of the host and growth inhibition of the parasite will be discussed on the basis of the present results.