Cathepsins L and B in Dysdercus peruvianus, Rhodnius prolixus, and Mahanarva fimbriolata. Looking for enzyme adaptations to digestion.

Cysteine peptidases (CP) play a role as digestive enzymes in hemipterans similar to serine peptidases in most other insects. There are two major CPs: cathepsin L (CAL), which is an endopeptidase and cathepsin B (CAB) that is both an exopeptidase and a minor endopeptidase. There are thirteen putative CALs in Dysdercus peruvianus, which in some cases were confirmed by cloning their encoding genes. RNA-seq data showed that DpCAL5 is mainly expressed in the anterior midgut (AM), DpCAL10 in carcass (whole body less midgut), suggesting it is a lysosomal enzyme, and the other DpCALs are expressed in middle (MM) and posterior (PM) midgut. The expression data were confirmed by qPCR and enzyme secretion to midgut lumen by a proteomic approach. Two CAL activities were isolated by chromatography from midgut samples with similar kinetic properties toward small substrates. Docking analysis of a long peptide with several DpCALs modeled with digestive Tenebrio molitor CAL (TmCAL3) as template showed that on adapting to luminal digestion DpCALs (chiefly DpCAL5) changed in relation to their ancestral lysosomal enzyme (DpCAL10) mainly at its S2 subsite. A similar conclusion arrived from structure alignment-based clustering of DpCALs based on structural similarity of the modeled structures. Changes mostly on S2 subsite could mean the enzymes turn out less peptide-bond selective, as described in TmCALs. R. prolixus CALs changed on adapting to luminal digestion, although less than DpCALs. Both D. peruvianus and R. prolixus have two digestive CABs which are expressed in the same extension as CALs, in the first digestive section of the midgut, but less than in the other midgut sections. Mahanarva fimbriolata does not seem to have digestive CALs and their digestive CABs are mainly expressed in the first digestive section of the midgut and do not diverge much from their lysosomal counterparts. The data suggest that CABs are necessary at the initial stage of digestion in CP-dependent Hemipterans, which action is completed by CALs with low peptide-bond selectivity in Heteroptera species. In M. fimbriolata protein digestion is supposed to be associated with the inactivation of sap noxious proteins, making CAB sufficient as digestive CP. Hemipteran genomes and transcriptome data showed that CALs have been recruited as digestive enzymes only in heteropterans, whereas digestive CABs occur in all hemipterans.

Silencing of RpATG8 impairs the biogenesis of maternal autophagosomes in vitellogenic oocytes, but does not interrupt follicular atresia in the insect vector Rhodnius prolixus.

Follicular atresia is the mechanism by which the oocyte contents are degraded during oogenesis in response to stress conditions, allowing the energetic resources stored in the developing oocytes to be reallocated to optimize female fitness. Autophagy is a conserved intracellular degradation pathway where double-membrane vesicles are formed around target organelles leading to their degradation after lysosome fusion. The autophagy-related protein 8 (ATG8) is conjugated to the autophagic membrane and has a key role in the elongation and closure of the autophagosome. Here we identified one single isoform of ATG8 in the genome of the insect vector of Chagas Disease Rhodnius prolixus (RpATG8) and found that it is highly expressed in the ovary during vitellogenesis. Accordingly, autophagosomes were detected in the vitellogenic oocytes, as seen by immunoblotting and electron microscopy. To test if autophagosomes were important for follicular atresia, we silenced RpATG8 and elicited atresia in vitellogenic females by Zymosan-A injections. We found that silenced females were still able to trigger the same levels of follicle atresia, and that their atretic oocytes presented a characteristic morphology, with accumulated brown aggregates. Regardless of the difference in morphology, RpATG8-silenced atretic oocytes presented the same levels of protein, TAG and PolyP, as detected in control atretic oocytes, as well as the same levels of acidification of the yolk organelles. Because follicular atresia has the ultimate goal of restoring female fitness, we tested if RpATG8-silenced atresia would result in female physiology and behavior changes. Under insectarium conditions, we found that atresia-induced control and RpATG8-silenced females present no changes in blood meal digestion, survival, oviposition, TAG content in the fat body, haemolymph amino acid levels and overall locomotor activity. Altogether, we found that autophagosomes are formed during oogenesis and that the silencing of RpATG8 impairs autophagosome biogenesis in the oocytes. Nevertheless, regarding major macromolecule degradation and adaptations to the fitness costs imposed by triggering an immune response, we found that autophagic organelles are not essential for follicle atresia in R. prolixus.

Generalist host species drive Trypanosoma cruzi vector infection in oil palm plantations in the Orinoco region, Colombia.

BACKGROUND: Oil palm plantation establishment in Colombia has the potential to impact Chagas disease transmission by increasing the distribution range of Rhodnius prolixus. In fact, previous studies have reported Trypanosoma cruzi natural infection in R. prolixus captured in oil palms (Elaeis guineensis) in the Orinoco region, Colombia. The aim of this study is to understand T. cruzi infection in vectors in oil palm plantations relative to community composition and host dietary specialization by analyzing vector blood meals and comparing these results to vectors captured in a native palm tree species, Attalea butyracea. METHODS: Rhodnius prolixus nymphs (n = 316) were collected from A. butyracea and E. guineensis palms in Tauramena, Casanare, Colombia. Vector blood meals from these nymphs were determined by amplifying and sequencing a vertebrate-specific 12S rRNA gene fragment. RESULTS: Eighteen vertebrate species were identified and pigs (Sus scrofa) made up the highest proportion of blood meals in both habitats, followed by house mouse (Mus musculus) and opossum (Didelphis marsupialis). Individual bugs feeding only from generalist mammal species had the highest predicted vector infection rate, suggesting that generalist mammalian species are more competent hosts for T. cruzi infection . CONCLUSIONS: Oil palm plantations and A. butyracea palms found in altered areas provide a similar quality habitat for R. prolixus populations in terms of blood meal availability. Both habitats showed similarities in vector infection rate and potential host species, representing a single T. cruzi transmission scenario at the introduced oil palm plantation and native Attalea palm interface.

The involvement of Rhopr-CRF/DH in feeding and reproduction in the blood-gorging insect Rhodnius prolixus.

Rhodnius prolixus is a blood-gorging insect and a vector for human Chagas disease. The insect transmits the disease following feeding, when it excretes urine and feces contaminated with the Trypanosoma cruzi parasite. A corticotropin-releasing factor-like peptide acts as a diuretic hormone in R. prolixus (Rhopr-CRF/DH); however, its distribution throughout the insect's central nervous system (CNS) and the expression of its receptor in feeding-related tissue as well as the female reproductive system suggests a multifaceted role for the hormone beyond that of diuresis. Here we investigate the involvement of Rhopr-CRF/DH in feeding and reproduction in R. prolixus. Immunohistochemistry of the CNS showed diminished CRF-like staining in neurosecretory cells (NSCs) of the mesothoracic ganglionic mass (MTGM) immediately following feeding, and partial restocking of those same cells two hours later, indicating Rhopr-CRF/DH stores in this regions are involved in feeding. The results of the temporal qPCR analysis were consistent with the immunohistochemical findings, showing an increase in Rhopr-CRF/DH transcript expression in the MTGM immediately after feeding, presumably capturing the restocking of Rhopr-CRF/DH in the lateral NSCs following release of the peptide during feeding. Elevating haemolymph Rhopr-CRF/DH titres by injection of Rhopr-CRF/DH prior to feeding resulted in the intake of a significantly smaller blood meal in 5th instars and adults without an apparent effect on the rate of short-term diuresis. When adult females were injected with Rhopr-CRF/DH, they also produced and laid significantly fewer eggs. Finally, in vitro oviduct contraction assays illustrate that Rhopr-CRF/DH inhibits the amplitude of contractions of the lateral oviducts, highlighting a potential mechanism via which the hormone diminishes reproductive capacity. To conclude, the study of the Rhopr-CRF/DH pathway, its components and mechanisms of action, has implications for vector control by highlighting targets to alter feeding, diuresis, and reproduction of this disease vector.

[Blood-feeding of Rhodnius prolixus].

Triatomines (Hemiptera: Reduviidae) are blood-sucking insect vectors of the protozoan Trypanosoma cruzi which is the causative agent of Chagas' disease. Rhodnius prolixus is the most epidemiologically important vector of T. cruzi in Colombia. Triatomines are regarded to be vessel-feeders as they obtain their blood meals from vertebrate hosts by directly inserting their mouthparts into vessels. Microscopic techniques are useful for visualizing and describing the morphology of biological structures. Here, we show images of the blood-feeding of R. prolixus, including some histological features by light microscopy and scanning electron microscopy of the mouthparts of R. prolixus when feeding on a laboratory mouse.

Sympatry influence in the interaction of Trypanosoma cruzi with triatomine

Abstract INTRODUCTION: Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely distributed in nature, circulating between triatomine bugs and sylvatic mammals, and has large genetic diversity. Both the vector species and the genetic lineages of T. cruzi present a varied geographical distribution. This study aimed to verify the influence of sympatry in the interaction of T. cruzi with triatomines. Methods: The behavior of the strains PR2256 (T. cruzi II) and AM14 (T. cruzi IV) was studied in Triatoma sordida (TS) and Rhodnius robustus (RR). Eleven fifth-stage nymphs were fed by artificial xenodiagnosis with 5.6 × 103 blood trypomastigotes/0.1mL of each T. cruzi strain. Every 20 days, their excreta were examined for up to 100 days, and every 30 days, the intestinal content was examined for up to 120 days, by parasitological (fresh examination and differential count with Giemsa-stained smears) and molecular (PCR) methods. Rates of infectivity, metacyclogenesis and mortality, and mean number of parasites per insect and of excreted parasites were determined. RESULTS: Sympatric groups RR+AM14 and TS+PR2256 showed higher values of the four parameters, except for mortality rate, which was higher (27.3%) in the TS+AM14 group. General infectivity was 72.7%, which was mainly proven by PCR, showing the following decreasing order: RR+AM14 (100%), TS+PR2256 (81.8%), RR+PR2256 (72.7%) and TS+AM14 (36.4%). CONCLUSIONS: Our working hypothesis was confirmed once higher infectivity and vector capacity (flagellate production and elimination of infective metacyclic forms) were recorded in the groups that contained sympatric T. cruzi lineages and triatomine species.

Metabolism and gas exchange patterns in Rhodnius prolixus.

Insect's metabolic rate and patterns of gas-exchange varies according to different factors such as: species, activity, mass, and temperature among others. One particular striking pattern of gas-exchange in insects is discontinuous gas-exchange cycles, for which many different hypotheses regarding their evolution have been stated. This article does not pretend to be an extensive review on the subject, rather to focus on the work performed on the haematophagous bug Rhodnius prolixus, a model organism used from the mid XX century until present days, with the great influence of Wigglesworth and his students/collaborator's work. I have no doubt that the renovated field of insect gas-exchange has a bright future and will advance at large gaits thank to the help of this model organism, R. prolixus, whose entire genome has recently being unraveled.

Genes encoding defensins of important Chagas disease vectors used for phylogenetic studies.

Insects possess both cellular and humoral immune responses. The latter makes them capable to recognize and control invading pathogens after synthesis of a variety of small proteins, also known as antimicrobial peptides. Defensins, cysteine-rich cationic peptides with major activity against Gram-positive bacteria, are one ubiquitous class of antimicrobial peptides, widely distributed in different animal and plant taxa. Regarding triatomines in each of the so far analyzed species, various defensin gene isoforms have been identified. In the present study, these genes were sequenced and used as a molecular marker for phylogenetic analysis. Considering the vectors of Chagas disease the authors are reporting for the first time the presence of these genes in Triatoma sordida (Stål, 1859), Rhodnius nasutus (Stål, 1859), and Panstrongylus megistus (Burmeister, 1835). Members of the Triatoma brasiliensis species complex were included into the study to verify the genetic variability within these taxa. Mainly in their mature peptide, the deduced defensin amino acid sequences were highly conserved. In the dendrogram based on defensin encoding nucleotide, sequences the Triatoma Def3/4 genes were separated from the rest. In the dendrogram based on deduced amino acid sequences the Triatoma Def2/3/4 together with Rhodnius DefA/B pre-propeptides were separated from the rest. In the sub-branches of both the DNA and amino acid dendrograms, the genus Triatoma was separated from the genus Rhodnius as well as from P. megistus.

Repellence produced by monoterpenes on Rhodnius prolixus (Hemiptera: Reduviidae) decreases after continuous exposure to these compounds.

Botanical monoterpenes are secondary metabolites present in essential oils produced by plants. Some of them are insect repellents. The bloodsucking bug Rhodnius prolixus Ståhl (Hemiptera: Reduviidae) is one of the main vectors of Chagas disease in the north of South America and some countries in Central America. In this study, we studied the repellence produced by two monoterpenes, menthyl acetate and geraniol, on fifth instar nymphs of R. prolixus. In the absence of other stimuli, both menthyl acetate and geraniol produced a repellent effect from 740 µg/cm(2) and 74 µg/cm(2), respectively. Pre-exposure to each monoterpene reduced the repellent activity produced by the same substance. Additionally, pre-exposure to one monoterpene decreased the behavioral response of the nymphs to the other one. The repellent effect of both monoterpenes also decreased when nymphs' antennae were previously treated with the nitric oxide donor S-nitroso-N-acetyl-cysteine.

The exotic palm Roystonea oleracea (Jacq.) O.F. Cook as a rural biotype for Rhodnius neglectus Lent, 1954, in Caçu, State of Goiás

Introduction Rhodnius neglectus is a triatomine that colonizes different palm species. In this study, we aimed to describe the presence of this triatomine bug in the royal palms (Roystonea oleracea) in a rural region of the State of Goiás. Methods Palm infestation was investigated by dissecting the palms or by using live-bait traps. Results Two palm trees were infested by R. neglectus negative for Trypanosoma cruzi, the etiological agent for Chagas disease. In the study area, R. neglectus is frequently found in households. Conclusions The adaptation of this species to palm trees introduced in Brazil for landscaping purposes poses another challenge for controlling the vectors of Chagas disease. .

Temperature-dependent variation in gas exchange patterns and spiracular control in Rhodnius prolixus.

Insects display an array of respiratory behaviors, including the use of discontinuous gas exchange. This pattern is characterized by periods of spiracular closure, micro-openings (flutter), and complete openings during which the majority of gas exchange takes place. A current model of insect spiracular control suggests that spiracles are controlled by two interacting feedback loops, which produce the discontinuous pattern. The flutter period is thought to be initiated by a critically low partial pressure of oxygen, while the open period is initiated by a critically high CO2 threshold. The goal of our study was to test this control model under conditions of feeding-induced or temperature-induced changes in metabolic rate. We manipulated the metabolic rate of the insect Rhodnius prolixus using two discrete mechanisms: (1) feeding the insects a bloodmeal or (2) exposing them to a range of temperatures (18-38°C). Examining the variation in the gas exchange patterns produced by insects in each of these treatments allowed us to determine whether spiracular control is sensitive to metabolic rate and/or temperature. We found that increases in temperature caused significant decreases in open phase burst volumes and premature abandonment of discontinuous gas exchange cycles. These effects were not observed in fed individuals maintained at a single temperature despite their higher metabolic rates. Our results indicate that some part of the spiracular control mechanism is temperature sensitive, suggesting a possible role for pH in CO2 sensing.

Differential effects of ambient temperature on warm cell responses to infrared radiation in the bloodsucking bug Rhodnius prolixus.

Thermoreceptors provide animals with background information about the thermal environment, which is at least indirectly a prerequisite for thermoregulation and assists bloodsucking insects in the search for their host. Recordings from peg-in-pit sensilla and tapered hairs on the antennae of the bug Rhodnius prolixus revealed two physiologically different types of warm cells. Both types responded more strongly to temperature pulses produced by switching between two air streams at different constant temperatures than to infrared radiation pulses employed in still air. In addition, both warm cells were better able to discriminate small changes in air temperature than in infrared radiation. As convective and radiant heat determines the discharge, it is impossible for a single warm cell to signal the nature of the stimulus unequivocally. Individual responses are ambiguous, not with regard to temperature change, but with regard to its source. We argue that the bugs use mechanical flow information to differentiate between pulses of convective and radiant heat. However, if pulses of radiant heat occur together with a constant temperature air stream, the mechanical cues would not allow avoiding ambiguity that convective heat introduces into radiant heat stimulation. In this situation, the warm cell in the tapered hairs produced stronger responses than those in the peg-in-pit sensilla. The reversal in the excitability of the two types of warm cells provides a criterion by which to distinguish the combination of convective and radiant heat from the stimuli presented alone.

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.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Comportamiento de Rhodnius robustusLarousse,1927 (Hemiptera, Reduviidae) durante su alimentaciónen condiciones de laboratorio / Feeding behavior of Rhodnius robustus Larouse, 1927 (Hemiptera, Reduviidae) under laboratory conditions

Introducción.A pesar del control de los insectos domiciliados, se siguen presentando casos de enfermedad de Chagas con la participación en la transmisión de especies peridomiciliarias y selváticas, una de las cuales es Rhodnius robustus, especie reportada con infecciones naturales de Trypanosoma cruzi y T. rangeli. Objetivo. Describir el comportamiento de R. robustus durante su alimentación en condiciones de laboratorio. Materiales y métodos. En cada estadio de desarrollo de R. robustus se analizó el tiempo promedio para alcanzar la repleción, número promedio de deyecciones, porcentaje de individuos que defecan durante la comida y ganancia de peso después de la alimentación. Resultados. Rhodnius robustus tardó entre 19:26 y 44:55 (minutos:segundos) en alimentarse a repleción; los insectos defecaron en todos sus diferentes estadios durante la alimentación. El porcentaje de individuos que defecan durante la alimentación varía de 13,3 % a 93,3 %. Por otro lado, el consumo promedio de sangre en el quinto estadio, 337,19 mg, duplica la cantidad ingerida por los adultos, 161,25 mg. Conclusiones. En todos los estadios de R. robustus hay defecación durante la alimentación, factor que favorece su competencia vectorial. Sin embargo, el tiempo para la primera deyección, el porcentaje de deyecciones y los individuos que defecan durante la comida, así como la cantidad de sangre ingerida, varían entre los estadios, lo cual indica que el comportamiento alimenticio está mediado por variables dependientes de la edad/estadio.

Introduction: Despite of indoor insect control measures, American trypanosomiasis cases still occur, with the participation of both peridomestic and sylvan species. One of these species is Rhodnius robustus, which has been found naturally infected with Trypanosoma cruzi and Trypanosoma rangeli. Objective: To describe the feeding behavior of R. robustus under laboratory conditions. Materials and methods: We analyzed time spent for repletion, the number of defecations, the percentage and number of times insects defecated during blood intake, and the weight gained after blood feeding in each nymph stage of R. robustus. Results: Repletion for R. robustus took between 19:26 to 44:55 (minutes:seconds) and all nymph stages defecated during blood feeding. The proportion of individuals which defecated during blood feeding varied between 13.3% and 93.3%. On the other hand, the highest average blood intake was observed in the fifth stage (337.19 mg), which is twice the blood intake of adults (161.25 mg). Conclusions: All stages of R. robustus dejected during blood feeding, which is an important factor for its vectorial competence. Nevertheless, the time prior to the first dejection, the percentage of dejections and number of individuals that dejected upon blood feeding, as well as the amount of ingested blood varied among stages, which means that feeding behavior is mediated by variables depending on age/stage.

Recent experiments towards a model for fluid secretion in Rhodnius Upper Malpighian Tubules (UMT).

Three different methods have been used to improve a model for fluid secretion in Upper Malpighian Tubules (UMT) of the blood sucking insect Rhodnius prolixus. (I) In the first, UMT double perfusions in 5th instar Rhodnius were used to measure their fluid secretion rate. They were stimulated to secrete with 5-HT. Double perfusions allowed access separately to the basolateral and the apical cell membranes with pharmacological agents known to block different ion transport functions, namely ATPases, cotransporters and/or countertransporters and ion and water channels: ouabain, bafilomycin A1, furosemide, bumetanide, SITS, acetazolamide, amiloride, DPC, BaCl(2), pCMBS and DTT. The basic assumption is that changes in water movement reflect changes in ion transport mechanisms. (II) Intracellular Na(+) concentrations were measured with a fluorometric method in dissected R. prolixus UMT, under several experimental conditions. (III) ATPase activities were measured in R. prolixus UMT. A tentative model for the function of the UMT cell is presented. We find that (a) at the basolateral cell membrane, fundamental is a Na(+)-K(+)-2Cl(-) cotransporter; of intermediate importance are the Na(+)-K(+)-ATPase and a ouabain-insensitive Na(+)-ATPase, ion channels and Rp-MIP water channels. (b) At the apical cell membrane, most important are a V-H(+)-ATPase; and a K(+) and/or Na(+)-H(+) exchanger.

Biologia comparativa e comportamento alimentar de Rhodnius neglectus e Rhodnius robustus (Triatominae) sob condições de laboratório / Comparative biology and feeding behavior of Rhodnius neglectus and Rhodnius robustus (Triatominae) under laboratory conditions

INTRODUÇÃO: A competência vetorial de triatomíneos é determinada a partir de estudos sobre biologia e comportamento alimentar em condições de campo e/ou laboratório. Fatores como número de picadas, quantidade de sangue ingerido e tempo de defecação têm implicações na transmissão de tripanosomatídeos. Parâmetros biológicos e comportamentais de Rhodnius neglectus e R. robustus foram comparados experimentalmente para estimar diferenças no potencial de transmissão de tripanosomatídeos. MÉTODOS: Os triatomíneos foram observados diariamente para determinar o período de desenvolvimento ninfal, mortalidade, detecção da fonte alimentar, número de picadas, tempo do repasto sanguíneo, quantidade de sangue ingerido, tempo entre o final do repasto e a primeira defecação e frequência de defecação. RESULTADOS: Apesar do período ninfal de R. neglectus (156,4 ± 25,05d) ter sido menor que o de R. robustus (204,7 ± 13,22d), a mortalidade foi similar entre as espécies (63,8 e 65%, respectivamente). R. robustus e R. neglectus detectaram rapidamente a fonte alimentar, especialmente no primeiro estádio (2,5 e 1,6min, respectivamente). Apesar do tempo de repasto sanguíneo ter sido similar entre as espécies, R. robustus ingeriu em média uma maior quantidade de sangue em todos os estádios, com maiores valores para as ninfas V. As ninfas de R. neglectus picaram mais vezes as fontes alimentares, defecaram mais rápido e mais frequentemente que as de R. robustus. CONCLUSÕES: Sob as condições de laboratório usadas, R. neglectus possui um maior potencial para transmissão de Trypanosoma cruzi e T. rangeli que R. robustus, atributo que deve ser avaliado em infecções experimentais.

INTRODUCTION: The vector competence of triatomine insects is determined by studying their biology and feeding behavior under field and/or laboratory conditions. Factors including the number of bites, the amount of blood ingested and defecation time have implications for trypanosome transmission. The biological and behavioral parameters of Rhodnius neglectus and R. robustus were compared under experimental conditions to estimate differences in the potential transmission of trypanosomes. METHODS: The insects were observed daily to determine the period of nymphal development, mortality, detection of food source, number of bites, time of blood meal intake, amount of blood ingested, time elapsed between the end of the meal and the first defecation and the frequency of defecation. RESULTS: Although the nymphal development of R. neglectus (156.4 ± 25.05d) was lower than that of R. robustus (204.7 ± 13.22d), the mortality between species was similar (63.8 and 65% respectively).R. robustus and R. neglectus quickly located the food source, especially in the first instar (2.5 and 1.6 min, respectively). Although the time of blood meal intake was similar between the species, R. robustus ingested a larger amount of blood on average at all stages and exhibited higher values for the fifth instar. Nymphs of R. neglectus bit more frequently, and they defecated faster and more often than those of R. robustus. CONCLUSIONS: Under laboratory conditions, R. neglectus has a greater potential for transmission of Trypanosoma cruzi and T. rangeli than does R. robustus, an attribute that should be further evaluated in experimental infections.

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.

Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge.

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.