An updated catalog of lipocalins of the chagas disease vector Rhodnius prolixus (Hemiptera, Reduviidae).

The haematophagy process by arthropods has been one of the main targets of studies in the parasite-host interaction, and the kissing-bug Rhodnius prolixus, vector of the protozoan Trypanosoma cruzi, has been one of the main models for such studies. Still in the 1980s, it was identified that among the salivary proteins for disrupting vertebrate host homeostasis, lipocalins were among the most relevant proteins for this process. Since then, 30 lipocalins have been identified in the salivary glands of R. prolixus, that promotes vasodilatation, prevents inflammation, act as anticoagulants and inhibits platelet aggregation. The present work aims to identify new lipocalins from R. prolixus, combining transcriptome and genome data. Identified new genes were mapped and had their structure annotated. To infer an evolutionary relationship between lipocalins, and to support the predicted functions for each lipocalin, all amino acid sequences were used to construct phylogenetic trees. We identified a total of 29 new lipocalins, 3 new bioaminogenic-biding proteins (which act to inhibit platelet aggregation and vasodilation), 9 new inhibitors of platelet aggregation, 7 new apolipoproteins and 10 lipocalins with no putative function. In addition, we observed that several of the lipocalins are also expressed in different R. prolxius tissues, including gut, central nervous system, antennae, and reproductive organs. In addition to newly identified lipocalins and a mapping the new and old lipocalins in the genome of R. prolixus, our study also carried out a review on functional status and nomenclature of some of the already identified lipocalins. Our study reinforces that we are far from understanding the role of lipocalins in the physiology of R. prolixus, and that studies of this family are still of great relevance.

Salivary proteins electrophoretic patterns enabled differentiating Colombian Rhodnius Trans-Andean and Cis-Andean groups / [Los patrones electroforéticos de proteínas salivales permiten diferenciar los grupos transandino y cisandino de las especies de Rhodnius de Colombia].

Introduction: Rhodnius (Hemiptera: Reduviidae: Triatominae) species are made up of haematophagous insect vectors for Trypanosoma cruzi (Chagas' disease aetiological agent) and T. rangeli, an infective parasite that is not pathogenic for vertebrate hosts. The study of their salivary protein diversity enables the obtention of characteristic one-dimensional electrophoretic profiles of some triatomine species; however, few reports have dealt with Rhodnius species salivary proteins electrophoretic patterns. Objective: To compare R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus' salivary proteins one-dimensional electrophoretic profiles. Materials and methods: SDS-PAGE was used for obtaining electrophoretic profiles of saliva from the species under study. The unweighted pair group method with arithmetic mean (UPGMA) was used for constructing a phenogram. Results: Electrophoretic profiles of soluble saliva had protein bands ranging from 15 to 45 kDa, thereby enabling the five species studied to be differentiated. The phenogram revealed two main groups, one formed by the Pictipes and Prolixus cis-Andean groups and another consisting of the Pallescens trans-Andean group. Conclusion: Differences were revealed regarding R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus electrophoretic profiles of salivary proteins; their variability facilitated constructing a phenogram which was taxonomically congruent with the groups from the genus Rhodnius.

Introducción. Las especies Rhodnius (Hemiptera: Reduviidae: Triatominae) están conformadas por insectos hematófagos vectores de Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, y T. rangeli, parásito infectivo pero no patógeno para el vertebrado. El estudio de la diversidad proteica de la saliva de estos insectos permite la obtención de perfiles electroforéticos unidimensionales característicos de algunas especies de triatominos. Sin embargo, el reporte de los patrones electroforéticos de proteínas salivales de las especies de Rhodnius ha sido escaso. Objetivo. Hacer un análisis comparativo de los perfiles electroforéticos unidimensionales de las proteínas salivales de R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus. Materiales y métodos. Se obtuvieron los perfiles electroforéticos de la saliva de las especies en estudio mediante electroforesis en gel de poliacrilamida con dodecilsulfatosódico (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) y se construyó un fenograma mediante el método UPGMA (Unweighted Pair Group Method Using Arithmetic Averages). Resultados. Los perfiles electroforéticos de las proteínas solubles de saliva presentaron bandas en un rango de masa aproximado de 15 a 45 kDa, los cuales permitieron diferenciar las cinco especies estudiadas. El fenograma reveló la existencia de dos grupos principales: uno conformado por los grupos cisandinos Pictipes y Prolixus y otro constituido por el grupo transandino Pallescens. Conclusiones. Existen diferencias en los perfiles electroforéticos de las proteínas salivales entre R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus, cuya variabilidad permitió construir un fenograma congruente con los grupos del género Rhodnius.

The juvenile hormone described in Rhodnius prolixus by Wigglesworth is juvenile hormone III skipped bisepoxide.

Juvenile hormones (JHs) are sesquiterpenoids synthesized by the corpora allata (CA). They play critical roles during insect development and reproduction. The first JH was described in 1934 as a "metamorphosis inhibitory hormone" in Rhodnius prolixus by Sir Vincent B. Wigglesworth. Remarkably, in spite of the importance of R. prolixus as vectors of Chagas disease and model organisms in insect physiology, the original JH that Wigglesworth described for the kissing-bug R. prolixus remained unidentified. We employed liquid chromatography mass spectrometry to search for the JH homologs present in the hemolymph of fourth instar nymphs of R. prolixus. Wigglesworth's original JH is the JH III skipped bisepoxide (JHSB3), a homolog identified in other heteropteran species. Changes in the titer of JHSB3 were studied during the 10-day long molting cycle of 4th instar nymph, between a blood meal and the ecdysis to 5th instar. In addition we measured the changes of mRNA levels in the CA for the 13 enzymes of the JH biosynthetic pathway during the molting cycle of 4th instar. Almost 90 years after the first descriptions of the role of JH in insects, this study finally reveals that the specific JH homolog responsible for Wigglesworth's original observations is JHSB3.

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection.

Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite Trypanosoma cruzi, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total T. cruzi parasite counts in R. prolixus were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

Posible transmisión oral de la enfermedad de Chagas en trabajadores del sector de los hidrocarburos en Casanare, Colombia, 2014 / Possible oral transmission of Chagas disease among hydrocarbons sector workers in Casanare, Colombia, 2014

RESUMEN Introducción. Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, puede transmitirse por vía oral tras la ingestión de alimentos o bebidas contaminadas. En la semana epidemiológica 14 del 2014, se notificaron dos casos de enfermedad aguda de Chagas en Paz de Ariporo, Casanare, entre trabajadores del sector de los hidrocarburos, episodio que motivó la investigación epidemiológica en el área. Objetivo. Caracterizar la población afectada, establecer medidas de control y confirmar el brote. Materiales y métodos. Se hizo una investigación de brote con los siguientes componentes: a) búsqueda de personas sintomáticas (cuadro clínico sugerente de enfermedad de Chagas según la definición de caso), para remitirlas a atención médica; b) aplicación de una encuesta entomológica en 192 de 197 viviendas; c) inspección sanitaria y análisis microbiológico de muestras de alimentos, y d) estudio de reservorios. La organización y el análisis de los datos se hicieron mediante estadística descriptiva con el programa Epi-Info 7.1.5. Asimismo, se establecieron los índices de infestación en el domicilio y el peridomicilio. Resultados. Se registraron 552 personas expuestas y se confirmaron por laboratorio 40 casos (7,2 %); siete casos se dieron en mujeres (17,5 %) y 33 en hombres (82,5%), es decir, en una relación de 1:5. La edad promedio fue de 39,1 (± 10,8) años, la tasa de ataque, de 7,2 %, y la letalidad, de 5 % (2/40). Los signos y síntomas incluyeron fiebre en el 100 % de los casos, cefalea en el 80 %, mialgias y artralgias en el 65 %, edema facial en el 55 %, y dolor abdominal en el 37,5 %. El tiempo promedio de incubación fue de 17 (3-21) días. El índice de infestación de Rhodnius prolixus fue de 3,3 % en el domicilio y de 2,2 % en el peridomicilio. En los cinco restaurantes inspeccionados, se encontraron condiciones sanitarias deficientes y alimentos con niveles de contaminación microbiológica inaceptables. Por último, un perro y dos zarigüeyas fueron positivos para los anticuerpos IgG en la prueba ELISA. Conclusiones. Mediante el análisis de las características epidemiológicas, ambientales y sanitarias, se confirmó un brote agudo de enfermedad de Chagas por exposición ocupacional y de posible transmisión oral, que podría ser el de mayor proporción reportado hasta la fecha en Colombia.

ABSTRACT Introduction: Trypanosoma cruzi, the etiological agent for Chagas disease, can be transmitted by oral intake of contaminated food or drinks. During epidemiological week 14 of 2014, two cases of acute Chagas disease were notified among hydrocarbons sector workers in Paz de Ariporo, Casanare. Objective: To characterize the affected population, to establish control and prevention measures and to confirm the outbreak. Materials and methods: We conducted an outbreak investigation that included the following components: a) Search for symptomatic people compatible with Chagas disease according to the case definition for their referral to medical services; b) entomological survey (192/197 houses); c) sanitary inspection and microbiological analysis of food samples; and d) study of reservoirs. Data management and analysis were done with Epi-Info 7.1.5 using descriptive statistics. We also calculated intradomicile and peridomicile triatomine infestation indexes. Results: We detected 552 exposed people; 40 had the disease (7.2%), of whom seven were women (17,5%) and 33, men (82.5%), i.e., a male-female ratio of 5:1. The mean age was 39.1 ± 10.8 years; the attack rate was 7.2% and lethality, 5% (2/40). Symptoms included fever (100% of cases), headache (80%), myalgia and arthralgia (65%), facial edema (55%), and abdominal pain (37.5%). The mean incubation time was 17 days (range: 3-21). Rhodnius prolixus domiciliary infestation index was 3.3 % and 2.2% in the peridomicile. In the five restaurants inspected sanitary conditions were deficient and food samples were microbiologically non-conforming. We found a dog and two opossums positive for IgG antibodies by ELISA. Conclusions: Environmental, sanitary and epidemiological conditions at the place confirmed an outbreak of Chagas diseases related to occupational exposure, possibly by oral transmission, which may be the largest to date in Colombia.

[Diversity of Triatominae (Hemiptera: Reduviidae) in Santander, Colombia: Epidemiological implications].

INTRODUCTION: Domestic and wild triatomines in the department of Santander have an epidemiological impact, as recently they have been linked to outbreaks of acute Chagas disease. The analysis of their diversity and temporal variation contributes to the understanding of their biology and ecology in one of the most endemic areas of the country. OBJECTIVES: To analyze triatominae diversity in two regions of Santander. MATERIALS AND METHODS: We analyzed the triatomine records for Santander contained in the CINTROP-UIS entomology lab database. We grouped the information for two regions: the Middle Magdalena area and the Andean region, and for each one we designed species accumulation and range-abundance curves, we calculated diversity and equality indices, and we analyzed colonization and temporal variation or persistence of the community. RESULTS: Ninety five percent of triatomines came from the Andean area and 4.57% from Magdalena Medio, with nine and ten species each. The dominant species in the Andean area were Rhodnius prolixus and Triatoma dimidiata while in Magdalena Medio they were Rhodnius pallescens and Panstrongylus geniculatus. We found a greater diversity and richness in Middle Magdalena compared to the Andean area. The temporal variation showed persistence of communities over time. CONCLUSIONS: Results revealed differences in the diversity of the two regions and the potential of wild species to occupy artificial ecotopes. Triatomines intrusion and the recent involvement of wild species in the transmission of Trypanosoma cruzi emphasize the need to further investigate the ecology of these vectors in order to guide population control strategies.

[Risk of transmission of Chagas disease by intrusion of triatomines and wild mammals in Bucaramanga, Santander, Colombia].

INTRODUCTION: Notice of triatomines in dwellings of some neighborhoods in Bucaramanga motivated the realization of this study.Objetive: To evaluate the intrusion of triatomines and mammals, as well as some risk factors in urban dwellings. MATERIALS AND METHODS: Triatomines were collected in a neighborhood in Bucaramanga, Santander, on a monthly basis during one year with participation of the community. Collection included manual search in lamp posts, use of light traps, animal bait, and chemical attractants in nearby forests. Reservoirs were collected with bait traps. Insects and mammals were identified and examined in order to determine their natural infection. Risk factors in homes were assessed by means of a social-environmental survey. RESULTS: Eleven adult specimens of Pastrongylus geniculatus, as well as 63 of Rhodnius pallescens were collected in the forest, recreational peridomiciliary areas, and houses. Even two females and 21 nymphs of R. pallescens were found in bedrooms. Two specimens of Didelphis marsupialis were captured in neighboring forests. Out of the eleven P. geniculatus captured, nine were examined. Of these, five were positive for Trypanosoma cruzi. It was not possible to establish a significant risk factor;however, the dwellings with report of triatomines were located nearer to the adjacent forest. CONCLUSIONS: The finding of intrusive triatominae species and mammals with T. cruzi in intradomiciliary and peridomiciliary areas and periurban forests prove the potential risk to acquire infection from these populations that dwell in urban housing adjacent to these ecotopes where the sylvan cycle is kept.

[Possible oral transmission of Chagas disease among hydrocarbons sector workers in Casanare, Colombia, 2014].

INTRODUCTION: Trypanosoma cruzi, the etiological agent for Chagas disease, can be transmitted by oral intake of contaminated food or drinks. During epidemiological week 14 of 2014, two cases of acute Chagas disease were notified among hydrocarbons sector workers in Paz de Ariporo, Casanare. OBJECTIVE: To characterize the affected population, to establish control and prevention measures and to confirm the outbreak. MATERIALS AND METHODS: We conducted an outbreak investigation that included the following components: a) Search for symptomatic people compatible with Chagas disease according to the case definition for their referral to medical services; b) entomological survey (192/197 houses); c) sanitary inspection and microbiological analysis of food samples; and d) study of reservoirs. Data management and analysis were done with Epi-Info 7.1.5 using descriptive statistics. We also calculated intradomicile and peridomicile triatomine infestation indexes. RESULTS: We detected 552 exposed people; 40 had the disease (7.2%), of whom seven were women (17,5%) and 33, men (82.5%), i.e., a male-female ratio of 5:1. The mean age was 39.1 ± 10.8 years; the attack rate was 7.2% and lethality, 5% (2/40). Symptoms included fever (100% of cases), headache (80%), myalgia and arthralgia (65%), facial edema (55%), and abdominal pain (37.5%). The mean incubation time was 17 days (range: 3-21). Rhodnius prolixus domiciliary infestation index was 3.3 % and 2.2% in the peridomicile. In the five restaurants inspected sanitary conditions were deficient and food samples were microbiologically non-conforming. We found a dog and two opossums positive for IgG antibodies by ELISA. CONCLUSIONS: Environmental, sanitary and epidemiological conditions at the place confirmed an outbreak of Chagas diseases related to occupational exposure, possibly by oral transmission, which may be the largest to date in Colombia.

Diversidad de Triatominae (Hemiptera: Reduviidae) en Santander, Colombia: implicaciones epidemiológicas / Diversity of Triatominae (Hemiptera: Reduviidae) in Santander, Colombia: Epidemiological implications

Resumen Introducción: Los triatominos domiciliados y silvestres constituyen un problema de impacto epidemiológico en el departamento de Santander, pues se han asociado recientemente con brotes agudos de la enfermedad de Chagas, por lo cual el análisis de su diversidad y variación temporal contribuye al conocimiento de su biología y ecología en una de las áreas más endémicas del país. Objetivo: Analizar la diversidad de triatominos en dos regiones de Santander. Materiales y métodos: Se analizó la información de la base de datos del Laboratorio de Entomología del Centro de Investigaciones en Enfermedades Tropicales de la Universidad Industrial de Santander (CINTROP-UIS), la cual contiene registros de triatominos en Santander. La información se separó en dos regiones, el Magdalena Medio y la zona andina, para cada una de las cuales se diseñaron curvas de acumulación de especies y de rango de abundancia, se calcularon los índices de diversidad y de igualdad, se analizó la colonización y se evaluó la variación temporal o persistencia de la comunidad. Resultados: El 95 % de los triatominos provenía de la zona andina y, el 4,57 %, del Magdalena Medio, con nueve y diez especies, respectivamente. Se encontró mayor diversidad y riqueza en el Magdalena Medio en comparación con la zona andina. Las especies dominantes en la zona andina fueron Rhodnius prolixus y Triatoma dimidiata, mientras que en Magdalena Medio fueron Rhodnius pallescens y Panstrongylus geniculatus. La variación temporal mostró persistencia de las comunidades en el tiempo. Conclusiones:. Los resultados evidenciaron diferencias en la diversidad de las dos regiones, además del potencial de las especies silvestres para ocupar ecótopos artificiales. La intrusión de triatominos y la reciente incriminación de especies silvestres en la transmisión de Trypanosoma cruzi, indican la necesidad de un mayor conocimiento de la ecología de estos vectores para orientar las estrategias de control.

Abstract Introduction: Domestic and wild triatomines in the department of Santander have an epidemiological impact, as recently they have been linked to outbreaks of acute Chagas disease. The analysis of their diversity and temporal variation contributes to the understanding of their biology and ecology in one of the most endemic areas of the country. Objectives: To analyze triatominae diversity in two regions of Santander. Materials and methods: We analyzed the triatomine records for Santander contained in the CINTROPUIS entomology lab database. We grouped the information for two regions: the Middle Magdalena area and the Andean region, and for each one we designed species accumulation and range-abundance curves, we calculated diversity and equality indices, and we analyzed colonization and temporal variation or persistence of the community. Results: Ninety five percent of triatomines came from the Andean area and 4.57% from Magdalena Medio, with nine and ten species each. The dominant species in the Andean area were Rhodnius prolixus and Triatoma dimidiata while in Magdalena Medio they were Rhodnius pallescens and Panstrongylus geniculatus. We found a greater diversity and richness in Middle Magdalena compared to the Andean area. The temporal variation showed persistence of communities over time. Conclusions: Results revealed differences in the diversity of the two regions and the potential of wild species to occupy artificial ecotopes. Triatomines intrusion and the recent involvement of wild species in the transmission of Trypanosoma cruzi emphasize the need to further investigate the ecology of these vectors in order to guide population control strategies.

Riesgo de transmisión de la enfermedad de Chagas por intrusión de triatominos y mamíferos silvestres en Bucaramanga, Santander, Colombia / Risk of transmission of Chagas disease by intrusion of triatomines and wild mammals in Bucaramanga, Santander, Colombia

Introducción: La notificación de triatominos en las viviendas de algunos barrios de Bucaramanga motivó la realización de este estudio. Objetivo: Evaluar la intrusión de triatominos y mamíferos, así como algunos factores de riesgo para la enfermedad de Chagas en viviendas urbanas. Materiales y métodos: En un barrio de Bucaramanga, Santander, se recolectaron triatominos mensualmente durante un año con participación comunitaria mediante búsqueda manual en el alumbrado público, y el uso de trampas de luz, cebo animal y atrayentes químicos en el bosque cercano. Los reservorios se recolectaron con trampas cebadas. Los insectos y mamíferos se determinaron y examinaron para establecer su infección natural. Los factores de riesgo de las viviendas se midieron mediante una encuesta sobre factores sociales y ambientales. Resultados: Se recolectaron 11 adultos de Panstrongylus geniculatus y 63 de Rhodnius pallescens en el bosque, en zonas de recreación en el peridomicilio y en el domicilio, incluidas dos hembras y 21 ninfas de R. pallescens en dormitorios. Se capturaron dos ejemplares de Didelphis marsupialis en el bosques adyacente. De los 11 individuos de P. geniculatus capturados, se examinaron nueve, de los cuales cinco fueron positivos para Trypanosoma cruzi (56 %); de los 63 individuos de R. pallescens capturados, se examinaron ocho, cuatro de los cuales fueron positivos para T. cruzi (50 %). De dos especímenes de D. marsupiales capturados, uno fue examinado y se encontró que era positivo para T. cruzi. No se pudo establecer un factor de riesgo significativo, sin embargo, las viviendas con reporte de triatominos se encontraban más cerca del bosque adyacente. Conclusiones: El hallazgo de especies de triatominos intrusivas y de mamíferos con T. cruzi en el domicilio y el peridomicilio, así como en los bosques periurbanos, demuestra el riesgo de infección en las poblaciones que habitan en viviendas urbanas adyacentes a los ecótopos donde se mantiene el ciclo silvestre.

Abstract Introduction: Notice of triatomines in dwellings of some neighborhoods in Bucaramanga motivated the realization of this study. Objetive: To evaluate the intrusion of triatomines and mammals, as well as some risk factors in urban dwellings. Materials and methods: Triatomines were collected in a neighborhood in Bucaramanga, Santander, on a monthly basis during one year with participation of the community. Collection included manual search in lamp posts, use of light traps, animal bait, and chemical attractants in nearby forests. Reservoirs were collected with bait traps. Insects and mammals were identified and examined in order to determine their natural infection. Risk factors in homes were assessed by means of a social-environmental survey. Results: Eleven adult specimens of Pastrongylus geniculatus, as well as 63 of Rhodnius pallescens were collected in the forest, recreational peridomiciliary areas, and houses. Even two females and 21 nymphs of R. pallescens were found in bedrooms. Two specimens of Didelphis marsupialis were captured in neighboring forests. Out of the eleven P. geniculatus captured, nine were examined. Of these, five were positive for Trypanosoma cruzi. It was not possible to establish a significant risk factor; however, the dwellings with report of triatomines were located nearer to the adjacent forest. Conclusions: The finding of intrusive triatominae species and mammals with T. cruzi in intradomiciliary and peridomiciliary areas and periurban forests prove the potential risk to acquire infection from these populations that dwell in urban housing adjacent to these ecotopes where the sylvan cycle is kept.

Chitin is a component of the Rhodnius prolixus midgut.

Chitin is an essential component of the peritrophic matrix (PM), which is a structure that lines the insect's gut and protects against mechanical damage and pathogens. Rhodnius prolixus (Hemiptera: Reduviidae) does not have a PM, but it has an analogous structure, the perimicrovillar membrane (PMM); chitin has not been described in this structure. Here, we show that chitin is present in the R. prolixus midgut using several techniques. The FTIR spectrum of the KOH-resistant putative chitin-material extracted from the midgut bolus showed peaks characteristic of the chitin molecule at 3500, 1675 and 1085 cm(1). Both the midgut bolus material and the standard chitin NMR spectra showed a peak at 1.88 ppm, which is certainly due to methyl protons in the acetamide a group. The percentages of radioactive N-acetylglucosamine (CPM) incorporated were 2 and 4% for the entire intestine and bolus, respectively. The KOH-resistant putative chitin-material was also extracted and purified from the N-acetylglucosamine radioactive bolus, and the radioactivity was estimated through liquid scintillation. The intestinal CHS cDNA translated sequence was the same as previously described for the R. prolixus cuticle and ovaries. Phenotypic alterations were observed in the midgut of females with a silenced CHS gene after a blood meal, such as retarded blood meal digestion; the presence of fresh blood that remained red nine days after the blood meal; and reduced trachea and hemozoin content compared with the control. Wheat germ agglutinin (a specific probe that detects chitin) labeling proximal to the intestine (crop and midgut) was much lower in females with a silenced CHS gene, especially in the midgut region, where almost no fluorescence signal was detected compared with the control groups. Midguts from females with a CHS gene silenced by dsRNA-CHS and control midguts pre-treated with chitinase showed that the chitin-derived fluorescence signal decreased in the region around the epithelium, the region facing the midgut and projections towards the intestinal lumen when evaluated microscopically. The relative reduction in CHS transcripts by approximately 80% using an RNAi assay supports the phenotypical alterations in the midgut observed using fluorescence microscopy assays. These data show that chitin is present in the R. prolixus midgut epithelium and in its surface projections facing the lumen. The CHS gene expression and the presence of chitin in the R. prolixus midgut may suggest a target for controlling Chagas disease vectors and addressing this public health problem.

Molecular characterization of Rhodnius prolixus' embryonic cuticle.

The embryonic cuticle (EC) of Rhodnius prolixus envelopes the entire body of the embryo during hatching and provides physical protection, allowing the embryo to pass through a narrow chorionic border. Most of the knowledge about the EC of insects is derived from studies on ultrastructure and secretion processes during embryonic development, and little is known about the molecular composition of this structure. We performed a comprehensive molecular characterization of the major components extracted from the EC of R. prolixus, and we discuss the role of the different molecules that were identified during the eclosion process. The results showed that, similar to the post-embryonic cuticles of insects, the EC of R. prolixus is primarily composed of carbohydrates (57%), lipids (19%), and proteins (8%). Considering only the carbohydrates, chitin is by far the major component (approximately 70%), and it is found primarily along the body of the EC. It is scarce or absent in its prolongations, which are composed of glycosaminoglycans. In addition to chitin, we also identified amino (15%), neutral (12%) and acidic (3%) carbohydrates in the EC of R. prolixus. In addition carbohydrates, we also identified neutral lipids (64.12%) and phospholipids (35.88%). Proteomic analysis detected 68 proteins (55 were identified and 13 are hypothetical proteins) using the sequences in the R. prolixus genome (http://www.vectorbase.org). Among these proteins, 8 out of 15 are associated with cuticle metabolism. These proteins are unequivocally cuticle proteins, and they have been described in other insects. Approximately 35% of the total proteins identified were classified as having a structural function. Chitin-binding protein, amino peptidase, amino acid oxidase, oxidoreductase, catalase and peroxidase are all proteins associated with cuticle metabolism. Proteins known to be cuticle constituents may be related to the function of the EC in assisting the insect during eclosion. To our knowledge, this is the first study to describe the global molecular composition of an EC in insects.

In search of magnetosensitivity and ferromagnetic particles in Rhodnius prolixus: behavioral studies and vibrating sample magnetometry.

Magnetoreception is a sensory mechanism with wide phylogenetic distribution, which many organisms use for navigation and orientation. Radical pair reactions and the use of magnetic particles have been proposed as mechanisms for magnetosensitivity in terrestrial animals. Magnetosensitivity and the presence of a ferromagnetic material were tested in the hematophagous bug Rhodnius prolixus (Hemiptera: Reduviidae: Triatominae) vector of Chagas disease in Colombia and Venezuela. R. prolixus is well known in both countries for its active dispersal that allows flow of individuals from sylvatic to domestic environments. Behavioral experiments quantifying the number of body rotations and quadrant changes in a Petri dish were carried out, applying 1 mT artificial field in a constant direction for 45 min and rotated 180° every 5 min for 45 min. In addition, magnetite presence in the abdomens of Apis mellifera (positive control) and the bodies of R. prolixus was tested using a vibrating sample magnetometer (VSM). No differences in the number of body rotations and quadrant changes were found in R. prolixus with and without the presence of an artificial magnetic field. Results obtained with the VSM indicate presence of ferromagnetic material (hysteresis loop) in A. mellifera abdomens and absence of ferromagnetic material in R. prolixus bodies. Both VSM and behavioral results suggest that magnetosensitivity by a ferromagnetic hypothesis is not present in R. prolixus. Finally, our results indicate that the VSM magnetometer is a sensitive technique for detecting ferromagnetic material in insect tissues.

Rhodnius prolixus lipophorin: lipid composition and effect of high temperature on physiological role.

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin-layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55 °C. When it was pretreated at 75 °C, maximal inhibition of phospholipid transfer was observed after 3-min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75 °C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55 °C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75 °C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65 °C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85 °C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.

OKB, a novel family of brain-gut neuropeptides from insects.

In insects, neuropeptides play a central role in the control of most physiological processes. The knowledge and characterization of new neuropeptide families, is of interest on the fields of Genetics, Genomics, Neurobiology, Endocrinology and Evolution. This knowledge also provides the tools for the design of peptidomimetics, pseudopeptides or small molecules, capable of disrupting the physiological processes regulated by the signaling molecules and their receptors. This is a promising target for a novel generation of insecticides. Using database searches, mass spectrometry and RACE-PCR, we identified a neuropeptide precursor transcript encoding a new family of insect neuropeptides in the hemipteran Rhodnius prolixus. We named this precursor Orcokinin B, because is originated by the alternative splicing of the Orcokinin gen. EST and genomic data suggests that Orcokinin B is expressed in the nervous system and gut from several insect species, with the exception of Drosophila sp. (Diptera) and Acyirthosiphon pisum (Hemiptera). Mass spectrometry and RT-PCR confirmed the expression of Orcokinin B in brain and anterior midgut of R. prolixus. Furthermore, we identified orthologues of this new family of peptides in genomic and EST databases from Arachnids and Crustaceans.

Diversity of anti-haemostatic proteins in the salivary glands of Rhodnius species transmitters of Chagas disease in the greater Amazon.

The triatomines in the tribe Rhodniini are the main vectors of the Trypanosoma cruzi to humans in recent outbreaks of acute Chagas disease in the Amazon. These insects dwelling in palm trees do not colonize the human domicile. Their success to transmit the infection relies partially on the efficacy of their salivary gland apparatuses. Here we show the transcriptome of the Rhodnius brethesi and Rhodnius robustus salivary glands, comprising 56 and 122 clusters, respectively. Approximately one third of these clusters are described for the first time. The LC-MS/MS analysis identified 123 and 111 proteins in R. brethesi and R. robustus sialome, respectively. Noteworthy, lipocalin platelet aggregation inhibitors, inositol polyphosphate 5-phosphatases, and Kazal domain proteins, which are essential for the insect's successful acquisition of blood meals, were found in our analysis. Moreover, glutathione S transferase and antigen-5, which play roles in the insect's defense and resistance against insecticide, were also observed.

2-DE-based proteomic investigation of the saliva of the Amazonian triatomine vectors of Chagas disease: Rhodnius brethesi and Rhodnius robustus.

The triatomine bugs are obligatory haematophagous organisms that act as vectors of Chagas disease by transmitting the protozoan Trypanosoma cruzi. Their feeding success is strongly related to salivary proteins that allow these insects to access blood by counteracting host haemostatic mechanisms. Proteomic studies were performed on saliva from the Amazonian triatomine bugs: Rhodnius brethesi and R. robustus, species epidemiologically relevant in the transmission of T. cruzi. Initially, salivary proteins were separated by two-dimensional gel electrophoresis (2-DE). The average number of spots of the R. brethesi and R. robustus saliva samples were 129 and 135, respectively. The 2-DE profiles were very similar between the two species. Identification of spots by peptide mass fingerprinting afforded limited efficiency, since very few species-specific salivary protein sequences are available in public sequence databases. Therefore, peptide fragmentation and de novo sequencing using a MALDI-TOF/TOF mass spectrometer were applied for similarity-driven identifications which generated very positive results. The data revealed mainly lipocalin-like proteins which promote blood feeding of these insects. The redundancy of saliva sequence identification suggested multiple isoforms caused by gene duplication followed by gene modification and/or post-translational modifications. In the first experimental assay, these proteins were predominantly phosphorylated, suggesting functional phosphoregulation of the lipocalins.

Neuropeptide precursor gene discovery in the Chagas disease vector Rhodnius prolixus.

We show a straightforward workflow combining homology search in Rhodnius prolixus genome sequence with cloning by rapid amplification of cDNA ends and mass spectrometry. We have identified 32 genes and their transcripts that encode a number of neuropeptide precursors leading to 194 putative peptides. We validated by mass spectrometry 82 of those predicted neuropeptides in the brain of R. prolixus to achieve the first comprehensive genomic, transcriptomic and neuropeptidomic analysis of an insect disease vector. Comparisons of available insect neuropeptide sequences revealed that the R. prolixus genome contains most of the conserved neuropeptides in insects, many of them displaying specific features at the sequence level. Some gene families reported here are identified for the first time in the order Hemiptera, a highly biodiverse group of insects that includes many human, animal and plant disease agents.

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.

[Electrophoretic patterns of salivary hemeproteins (nitrophorines) of Rhodnius colombiensis and R. prolixus (Hemiptera, Reduviidae, Triatominae)]. / Patrones electroforéticos de hemoproteínas salivares (nitroforinas) de Rhodnius colombiensis y Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae).

INTRODUCTION: Salivary hemeprotein electrophoresis in starch gels is a recent technique used for differentiation of Rhodnius species with great phenotypic similarity. Furthermore, populations of the same species can be differentiated from geographically separated locales. Of the 15 described Rhodnius species in Latin America, at least eight have been reported in Colombia. OBJECTIVE: To use the salivary hemeproteins electrophoresis for R. prolixus and R. colombiensis identification. These two species are phenotypically similar and have overlapping domestic and sylvatic cycles where they occur in the upper basin of the Magdalena river, Central Colombia. MATERIAL AND METHODS: The content of salivary glands of each insect was subjected to starch gel electrophoresis using glycine buffer, and the bands were revealed with 3,3',5,5'-tetramethylbenzidine. Band patterns were photographically recorded. RESULTS: Electrophoretic patterns of salivary hemeproteins of R. prolixus and R. colombiensis were able to unequivocally differentiate the two species. CONCLUSION: The usefulness of the starch gel technique for distinguishing between R. prolixus and R. colombiensis was demonstrated as an additional tool to the morphometric and molecular methods already in use for differentiation of these two species.