Global characterization of gene expression in the brain of starved immature Rhodnius prolixus.

BACKGROUND: Rhodnius prolixus is a vector of Chagas disease and has become a model organism to study physiology, behavior, and pathogen interaction. The publication of its genome allowed initiating a process of comparative characterization of the gene expression profiles of diverse organs exposed to varying conditions. Brain processes control the expression of behavior and, as such, mediate immediate adjustment to a changing environment, allowing organisms to maximize their chances to survive and reproduce. The expression of fundamental behavioral processes like feeding requires fine control in triatomines because they obtain their blood meals from potential predators. Therefore, the characterization of gene expression profiles of key components modulating behavior in brain processes, like those of neuropeptide precursors and their receptors, seems fundamental. Here we study global gene expression profiles in the brain of starved R. prolixus fifth instar nymphs by means of RNA sequencing (RNA-Seq). RESULTS: The expression of neuromodulatory genes such as those of precursors of neuropeptides, neurohormones, and their receptors; as well as the enzymes involved in the biosynthesis and processing of neuropeptides and biogenic amines were fully characterized. Other important gene targets such as neurotransmitter receptors, nuclear receptors, clock genes, sensory receptors, and takeouts genes were identified and their gene expression analyzed. CONCLUSION: We propose that the set of neuromodulatory-related genes highly expressed in the brain of starved R. prolixus nymphs deserves functional characterization to allow the subsequent development of tools targeting them for bug control. As the brain is a complex structure that presents functionally specialized areas, future studies should focus on characterizing gene expression profiles in target areas, e.g. mushroom bodies, to complement our current knowledge.

Trypanosoma rangeli infection increases the exposure and predation endured by Rhodnius prolixus.

Trypanosoma rangeli is a protozoan that infects triatomines and mammals in Latin America, sharing hosts with Trypanosoma cruzi, the etiological agent of Chagas disease. Trypanosoma rangeli does not cause disease to humans but is strongly pathogenic to its invertebrate hosts, increasing mortality rates and affecting bug development and reproductive success. We have previously shown that this parasite is also capable of inducing a general increase in the locomotory activity of its vector Rhodnius prolixus in the absence of host cues. In this work, we have evaluated whether infection impacts the insect­vertebrate host interaction. For this, T. rangeli-infected and uninfected R. prolixus nymphs were released in glass arenas offering single shelters. After a 3-day acclimatization, a caged mouse was introduced in each arena and shelter use and predation rates were evaluated. Trypanosoma rangeli infection affected all parameters analysed. A larger number of infected bugs was found outside shelters, both in the absence and presence of a host. Infected bugs also endured greater predation rates, probably because of an increased number of individuals that attempted to feed. Interestingly, mice that predated on infected bugs did not develop T. rangeli infection, suggesting that the oral route is not effective for these parasites, at least in our system. Finally, a smaller number of infected bugs succeeded in feeding in this context. We suggest that, although T. rangeli is not transmitted orally, an increase in the proportion of foraging individuals would promote greater parasite transmission rates through an increased frequency of very effective infected-bug bites.

Trypanosoma cruzi-infected Rhodnius prolixus endure increased predation facilitating parasite transmission to mammal hosts.

Triatomine bugs aggregate with conspecifics inside shelters during daylight hours. At dusk, they leave their refuges searching for hosts on which to blood feed. After finding a host, triatomines face the threat of being killed, because hosts often prey on them. As it is known that many parasites induce the predation of intermediate hosts to promote transmission, and that ingestion of Trypanosoma cruzi-infected bugs represents a very effective means for mammal infection, we hypothesized that trypanosomes induce infected bugs to take increased risk, and, as a consequence, be predated when approaching a host. Therefore, we evaluated whether the predation risk and predation rates endured by Rhodnius prolixus increase when infected with T. cruzi. Assays were performed in square glass arenas offering one central refuge to infected and uninfected 5th instar nymphs. A caged mouse was introduced in each arena after a three-day acclimation interval to activate sheltered insects and induce them to approach it. As hypothesized, a significantly higher proportion of infected insects was predated when compared with uninfected ones (36% and 19%, respectively). Indeed, T. cruzi-infected bugs took higher risk (Approximation Index = 0.642) when compared with healthy ones (Approximation Index = 0.302) and remained outside the shelters when the host was removed from the arena. Our results show that infection by T. cruzi induces bugs to assume higher risk and endure higher predation rates. We reveal a hitherto unknown trypanosome-vector interaction process that increases infected bug predation, promoting increased rates of robust oral transmission. The significant consequences of the mechanism revealed here make it a fundamental component for the resilient maintenance of sylvatic, peridomestic and domestic cycles.

Triatomine and Trypanosoma cruzi discrete typing units distribution in a semi-arid area of northeastern Brazil.

The occurrence of triatomine species, their bloodmeal sources and the discrete typing units (DTUs) of Trypanosoma cruzi isolated from them were determined in different municipalities of the state of Rio Grande do Norte, Brazil. Triatomine captures were carried out in the rural areas of 23 municipalities. The genotyping of T. cruzi isolates was performed using the mitochondrial cytochrome c oxidase subunit 2 (coii) gene, the D7 region of the 24Sα rDNA, and the spliced leader intergenic region (SL-IR). Five triatomine species were captured, and the most frequent was Triatoma brasiliensis (84.3%; 916/1086), which was found in 16 of the 23 municipalities surveyed, and infested all types of environment investigated. The TcI DTU was found in all mesoregions surveyed in 51.5% (17/33) of the culture-positive samples. In contrast, TcII (9.1%; 3/33) was detected in the Central mesoregion, while TcIII (27.3%; 9/33) was found in all mesoregions. The geographic distribution and spatial overlap of different DTUs was inferred using the superposition of the radius of occurrence of isolates and using ecological niche distribution modelling. Triatoma brasiliensis was found infected in all mesoregions and with all three T. cruzi DTUs, including mixed infections. With regard to bloodmeal sources, the DNA of rodents was found in triatomines infected with either TcI or TcIII, while that of domestic animals and humans was associated with both single and mixed infections. Our findings demonstrate that different DTUs of T. cruzi are widely dispersed among triatomines in our study area. The association of T. brasiliensis with several different mammalian hosts, as well as overlapping areas with different DTUs, suggests that this triatomine species may have an important role as a vector in both anthropic and sylvatic environments.

Co-existing locomotory activity and gene expression profiles in a kissing-bug vector of Chagas disease.

The triatomine bug Rhodnius prolixus is a main vector of Chagas disease, which affects several million people in Latin-America. These nocturnal insects spend most of their locomotory activity during the first hours of the scotophase searching for suitable hosts. In this study we used multivariate analysis to characterize spontaneous locomotory activity profiles presented by 5th instar nymphs. In addition, we investigated whether sex and the expression of the foraging (Rpfor) gene could modulate this behavioral trait. Hierarchical Clustering and Redundancy Analyses detected individuals with distinct locomotory profiles. In addition to a great variation in locomotory intensity, we found that a proportion of nymphs walked during unusual time intervals. Locomotory activity profiles were mostly affected by the cumulative activity expressed by the nymphs. These effects promoted by cumulative activity were in turn influenced by nymph sex. Sex and the Rpfor expression had a significant influence on the profiles, as well as in the levels of total activity. In conclusion, the locomotory profiles evinced by the multivariate analyses suggest the co-existence of different foraging strategies in bugs. Additionally, we report sex-specific effects on the locomotion patterns of 5th instar R. prolixus, which are apparently modulated by the differential expression of the Rpfor gene.

Knockout of the CCCH zinc finger protein TcZC3H31 blocks Trypanosoma cruzi differentiation into the infective metacyclic form.

In the protozoan parasite Trypanosoma cruzi - the causative agent of Chagas disease - gene expression control is mainly post-transcriptional, where RNA-binding proteins (RBPs) play a central role, by controlling mRNA stability, distribution and translation. A large variety of RBPs are encoded in the T. cruzi genome, including the CCCH-type zinc finger (CCCH ZnF) protein family, which is characterized by the presence of the C-X7/8-C-X5-C-X3-H (CCCH) motif. In the related parasite T. brucei, CCCH ZnF proteins have been shown to control key differentiation steps in the parasite's life cycle. However, little is known about the CCCH ZnF proteins in T. cruzi. We have worked on the generation of T. cruzi mutants for CCCH ZnF proteins in an effort to shed light on the functions of these proteins in this parasite. Here, we characterize the expression and function of the CCCH ZnF protein TcZC3H31 of T. cruzi. TcZC3H31 is almost exclusively expressed in epimastigotes and metacyclic trypomastigotes, the parasite forms found in the invertebrate host. Importantly, we show that the epimastigote form of the T. cruzi knockout for the TcZC3H31 gene (TcZC3H31 KO) is incapable, both in vitro and in vivo (in infected triatomine insects), to differentiate into the metacyclic trypomastigote form, which is responsible for infection transmission from vectors to humans. The epimastigote forms recovered from the excreta of insects infected with TcZC3H31 KO parasites do not have the typical epimastigote morphology, suggesting that parasites are arrested in a mid-differentiation step. Also, epimastigotes overexpressing TcZC3H31 differentiate into metacyclics more efficiently than wild-type epimastigotes, in vitro. These data suggest that TcZC3H31 is an essential positive regulator of T. cruzi differentiation into the human-infective metacyclic form.

Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell-derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long-term cultured epimastigotes: resistance to complement-mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up-regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host-parasite interactions, showing that rdEpi can be infective to the mammalian host.

Trypanosomes Modify the Behavior of Their Insect Hosts: Effects on Locomotion and on the Expression of a Related Gene.

BACKGROUND: As a result of evolution, the biology of triatomines must have been significantly adapted to accommodate trypanosome infection in a complex network of vector-vertebrate-parasite interactions. Arthropod-borne parasites have probably developed mechanisms, largely still unknown, to exploit the vector-vertebrate host interactions to ensure their transmission to suitable hosts. Triatomines exhibit a strong negative phototaxis and nocturnal activity, believed to be important for insect survival against its predators. METHODOLOGY/PRINCIPAL FINDINGS: In this study we quantified phototaxis and locomotion in starved fifth instar nymphs of Rhodnius prolixus infected with Trypanosoma cruzi or Trypanosoma rangeli. T. cruzi infection did not alter insect phototaxis, but induced an overall 20% decrease in the number of bug locomotory events. Furthermore, the significant differences induced by this parasite were concentrated at the beginning of the scotophase. Conversely, T. rangeli modified both behaviors, as it significantly decreased bug negative phototaxis, while it induced a 23% increase in the number of locomotory events in infected bugs. In this case, the significant effects were observed during the photophase. We also investigated the expression of Rpfor, the triatomine ortholog of the foraging gene known to modulate locomotion in other insects, and found a 4.8 fold increase for T. rangeli infected insects. CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time that trypanosome infection modulates the locomotory activity of the invertebrate host. T. rangeli infection seems to be more broadly effective, as besides affecting the intensity of locomotion this parasite also diminished negative phototaxis and the expression of a behavior-associated gene in the triatomine vector.

Atividade locomotora, uso de abrigos e predação em Rhodnius prolixus: alterações induzidas pela infecção por tripanosomatídeos

Rhodnius prolixus é um inseto hematófago considerado principal transmissor do Trypanosoma cruzi, agente etiológico da doença de Chagas, na Colômbia e Venezuela. Além do T. cruzi, ele pode transmitir o Trypanosoma rangeli a várias espécies de mamíferos, incluindo o homem. Durante o dia, estes insetos são encontrados no interior de abrigos, agregados com seus co-específicos em um estado de imobilidade denominado a cinese. Durante a noite, estes insetos apresentam um perfil de atividade locomotora com dois picos característicos: um na primeira metade da escotofase, relacionado com a procura por hospedeiros e parceiros sexuais, e outro, na interfase entre o fim da fase escura e a fotofase, direcionado à procura de refúgios.

Como seus hospedeiros são comumente vertebrados predadores de insetos, os triatomíneos podem ser mortos durante sua busca e o subsequente processo de alimentação. Desta maneira, o presente estudo avaliou se a infecção pelos tripanosomas acima citados altera o nível de atividade locomotora, os padrões de uso de abrigos e as taxas de predação sofridas por ninfas de R. prolixus. Além disso, avaliou-se a possibilidadede camundongos se infectarem ao ingerir ninfas infectadas com T. rangeli. Inicialmente, demonstrou-se que insetos infectados com T. cruzi apresentam uma diminuição nos níveis de atividade locomotora durante as primeiras horas da escotofase. Em contraste, a infecção por T. rangeli induziu a um aumento no nível de atividade locomotora durante praticamente todo o ciclo diário. Os padrões de uso de abrigos por ninfas infectadas com T. cruzi praticamente não foram alterados, porém esses insetos foram significativamente mais predados que insetos sadios. Trypanosoma rangeli modificou todos os parâmetros avaliados relacionados com o uso de abrigo. A infecção por este parasito aumentou a porcentagem de insetos que permaneceram fora dos abrigos, a exposição ao hospedeiro e consequentemente suas taxas de predação. Por fim, demonstrou-se que camundongos não se infectam com T. rangeli pela via oral. As alterações observadas, principalmente aquelas das taxas de predação, podem ter importantes implicações na transmissão de ambos os tripanosomatídeos na natureza, e são discutidas no presente trabalho

Atividade locomotora, uso de abrigos e predação em Rhodnius prolixus: alterações induzidas pela infecção por tripanosomatídeos / Locomotor activity, use of shelters and predation in Rhodnius prolixus: changes induced by infection with trypanosomatids

Rhodnius prolixus é um inseto hematófago considerado principal transmissor do Trypanosoma cruzi, agente etiológico da doença de Chagas, na Colômbia e Venezuela. Além do T. cruzi, ele pode transmitir o Trypanosoma rangeli a várias espécies de mamíferos, incluindo o homem. Durante o dia, estes insetos são encontrados no interior de abrigos, agregados com seus co-específicos em um estado de imobilidade denominado a cinese. Durante a noite, estes insetos apresentam um perfil de atividade locomotora com dois picos característicos: um na primeira metade da escotofase, relacionado com a procura por hospedeiros e parceiros sexuais, e outro, na interfase entre o fim da fase escura e a fotofase, direcionado à procura de refúgios...