Alterations in energy metabolism of Rhodnius prolixus induced by Trypanosoma rangeli infection.

Trypanosoma rangeli is a protozoan parasite that infects triatomines and mammals in the Americas, producing mixed infections with Trypanosoma cruzi, the etiological agent of Chagas disease. The former parasite is not pathogenic to humans, but has different levels of pathogenicity, as well as causing physiological and behavioral alterations, to its invertebrate hosts. In this study, we measured locomotory activity, and the glyceride accumulation profile in the hemolymph and fat body, as well as the expression of key genes related to triglyceride metabolism, of Rhodnius prolixus nymphs infected with T. rangeli. We found that the locomotory activity of the insects was correlated with the amount of triglycerides in the fat body. Infected nymphs had increased activity when starved, and also had an accumulation of glycerides in the fat body and hemolymph. These alterations were also associated with a higher expression of the diacylglycerol acyltransferase, lipophorin and lipophorin receptor genes in the fat body. We infer that T. rangeli is able to alter the energetic processes of its invertebrate host, in order to increase the availability of lipids to the parasite, which, in turn modifies the activity levels of the insect. These alterations are discussed with regard to their potential to increase the transmission rate of the parasite.

Rhodnius spp. infestation in palm trees and natural infection by Trypanosoma cruzi and Trypanosoma rangeli in periurban and rural areas of state of the Rondônia, in the Brazilian Amazon.

The state of Rondônia in the Brazilian Amazon is prone to diseases transmitted by insect vectors because of the environmental and population changes resulting from large hydroelectric projects and the expansion of agricultural and livestock industries. The first case of Chagas disease by vectorial transmission was recorded in 2019 in a rural area in Rondônia, reinforcing the need for entomological surveillance. Hence, our goal was to estimate the abundance of Rhodnius spp. in palm trees located in rural and periurban areas and in Brazil-Bolivia border regions, perform domiciliary searches, and check for possible associations between triatomines and the presence/absence of palm-inhabiting fauna and outdoor farming, domestic animals, and buildings. The sampling took place in five municipalities of Rondônia in 2014 (June to August) and 2015 (April to June). Triatomines were collected by active searches during the selective pruning of palm tree crowns. Domiciliary inspections lasted from 30 to 60 min. A set of captured triatomines was analyzed for Trypanosoma cruzi and T. rangeli infection by PCR. Overall, 496 insects were captured during sampling of 150 palms in rural areas and 150 in periurban areas. No triatomine was found during active searches of 59 dwelling either indoors or outdoors. The majority of triatomines caught in the palm trees were identified as Rhodnius robustus (98.6%), and seven specimens were R. pictipes. Triatomine infestation was observed in only 20% of the sampled palms (61/300) in the vicinity of 26/59 households. Nearly half of the infested palm trees had only one or two triatomines, and few palms presented more than 15 triatomines. The municipality of Buritis had the highest triatomine abundance and percentage of infested palms; however, the highest triatomine density per infested palm was observed in Alvorada D'Oeste, where a quarter of the palms were infested. Ants, arachnids, termites, reptiles, and rodents were frequently found in palm trees. Dogs were the predominant domestic animals in households, whereas hens and cattle were the main farming animals. Model estimates showed that the number of triatomines was affected by the presence of henhouses and varied strongly between localities. No relationships were detected between the average number of triatomines and palm fauna and/or palm height. Overall, approximately half of the triatomines were infected with T. cruzi (51.4%) and/or T. rangeli (47.2%), reinforcing the need for continuous entomological surveillance and implementation of community-based approaches because the Brazilian state of Rondônia borders areas experiencing reinfestation by domiciled species and potential colonization of animal shelters by triatomines.

Azadirachtin interferes with basal immunity and microbial homeostasis in the Rhodnius prolixus midgut.

Rhodnius prolixus is an insect vector of two flagellate parasites, Trypanosoma rangeli and Trypanosoma cruzi, the latter being the causative agent of Chagas disease in Latin America. The R. prolixus neuroendocrine system regulates the synthesis of the steroid hormone ecdysone, which is essential for not only development and molting but also insect immunity. Knowledge for how this modulates R. prolixus midgut immune responses is essential for understanding interactions between the vector, its parasites and symbiotic microbes. In the present work, we evaluated the effects of ecdysone inhibition on R. prolixus humoral immunity and homeostasis with its microbiota, using the triterpenoid natural product, azadirachtin. Our results demonstrated that azadirachtin promoted a fast and lasting inhibitory effect on expression of both RpRelish, a nuclear factor kappa B transcription factor (NF-kB) component of the IMD pathway, and several antimicrobial peptide (AMP) genes. On the other hand, RpDorsal, encoding the equivalent NF-kB transcription factor in the Toll pathway, and the defC AMP gene were upregulated later in azadirachtin treated insects. The treatment also impacted on proliferation of Serratia marcescens, an abundant commensal bacterium. The simultaneous administration of ecdysone and azadirachtin in R. prolixus blood meals counteracted the azadirachtin effects on insect molting and also on expression of RpRelish and AMPs genes. These results support the direct involvement of ecdysone in regulation of the IMD pathway in the Rhodnius prolixus gut.

Infecting Triatomines with Trypanosomes.

The infection of triatomines with trypanosomes can be performed with different forms of the parasite, and the procedure is important not only for vector-parasite interaction studies but also for maintaining the infectivity of parasite strains, which guarantees more realistic biological and molecular investigations. Here, I describe how to infect the vector Rhodnius prolixus, a model species, with two different species of Trypanosoma.

A darker chromatic variation of Rhodnius pallescens infected by specific genetic groups of Trypanosoma rangeli and Trypanosoma cruzi from Panama.

BACKGROUND: Rhodnius pallescens, the only species of this genus reported in Panama, has a wide geographical distribution and is associated with most cases of Chagas disease and human infections with Trypanosoma rangeli in this country. Thus far, no phenotypic variants of this triatomine have been registered. Similarly, genotyping of the trypanosomes that infect this vector has only been partially evaluated. RESULTS: A total of 347 specimens of R. pallescens were collected in Attalea butyracea palm trees located near a mountainous community of the district of Santa Fe, province of Veraguas. Bugs were slightly longer and had a darker coloration compared to that reported for this species. Infection rates for trypanosomes performed with three PCR analyses showed that 41.3% of the adult triatomines were positive for T. cruzi, 52.4% were positive for T. rangeli and 28.6% had mixed T. cruzi/T. rangeli infections. Based on cox2 analysis, TcI was the single T. cruzi discrete typing unit (DTU) detected, and a genetic variant of KP1(-)/lineage C was the only genetic group found for T. rangeli. CONCLUSIONS: A darker chromatic variation of R. pallescens predominates in a mountainous region of Panama. These triatomines show high trypanosome infection rates, especially with T. rangeli. Regarding T. rangeli genetic diversity, complementary studies using other molecular markers are necessary to better define its phylogenetic position.

Triatomine physiology in the context of trypanosome infection.

Triatomines are hematophagous insects that feed on the blood of vertebrates from different taxa, but can occasionally also take fluids from invertebrate hosts, including other insects. During the blood ingestion process, these insects can acquire diverse parasites that can later be transmitted to susceptible vertebrates if they complete their development inside bugs. Trypanosoma cruzi, the etiological agent of Chagas disease, and Trypanosoma rangeli are protozoan parasites transmitted by triatomines, the latter only transmitted by Rhodnius spp. The present work makes an extensive revision of studies evaluating triatomine-trypanosome interaction, with special focus on Rhodnius prolixus interacting with the two parasites. The sequences of events encompassing the development of these trypanosomes inside bugs and the consequent responses of insects to this infection, as well as many pathological effects produced by the parasites are discussed.

Rhodnius prolixus: from physiology by Wigglesworth to recent studies of immune system modulation by Trypanosoma cruzi and Trypanosoma rangeli.

This review is dedicated to the memory of Professor Sir Vincent B. Wigglesworth (VW) in recognition of his many pioneering contributions to insect physiology which, even today, form the basis of modern-day research in this field. Insects not only make vital contributions to our everyday lives by their roles in pollination, balancing eco-systems and provision of honey and silk products, but they are also outstanding models for studying the pathogenicity of microorganisms and the functioning of innate immunity in humans. In this overview, the immune system of the triatomine bug, Rhodnius prolixus, is considered which is most appropriate to this dedication as this insect species was the favourite subject of VW's research. Herein are described recent developments in knowledge of the functioning of the R. prolixus immune system. Thus, the roles of the cellular defences, such as phagocytosis and nodule formation, as well as the role of eicosanoids, ecdysone, antimicrobial peptides, reactive oxygen and nitrogen radicals, and the gut microbiota in the immune response of R. prolixus are described. The details of many of these were unknown to VW although his work gives indications of his awareness of the importance to R. prolixus of cellular immunity, antibacterial activity, prophenoloxidase and the gut microbiota. This description of R. prolixus immunity forms a backdrop to studies on the interaction of the parasitic flagellates, Trypanosoma cruzi and Trypanosoma rangeli, with the host defences of this important insect vector. These parasites remarkably utilize different strategies to avoid/modulate the triatomine immune response in order to survive in the extremely hostile host environments present in the vector gut and haemocoel. Much recent information has also been gleaned on the remarkable diversity of the immune system in the R. prolixus gut and its interaction with trypanosome parasites. This new data is reviewed and gaps in our knowledge of R. prolixus immunity are identified as subjects for future endeavours. Finally, the publication of the T. cruzi, T. rangeli and R. prolixus genomes, together with the use of modern molecular techniques, should lead to the enhanced identification of the determinants of infection derived from both the vector and the parasites which, in turn, could form targets for new molecular-based control strategies.

Temperature and parasite life-history are important modulators of the outcome of Trypanosoma rangeli-Rhodnius prolixus interactions.

Trypanosoma rangeli is a protozoan parasite, which does not cause disease in humans, although it can produce different levels of pathogenicity to triatomines, their invertebrate hosts. We tested whether infection imposed a temperature-dependent cost on triatomine fitness using T. rangeli with different life histories. Parasites cultured only in liver infusion tryptose medium (cultured) and parasites exposed to cyclical passages through mice and triatomines (passaged) were used. We held infected insects at four temperatures between 21 and 30 °C and measured T. rangeli growth in vitro at the same temperatures in parallel. Overall, T. rangeli infection induced negative effects on insect fitness. In the case of cultured infection, parasite effects were temperature-dependent. Intermoult period, mortality rates and ecdysis success were affected in those insects exposed to lower temperatures (21 and 24 °C). For passaged-infected insects, the effects were independent of temperature, intermoult period being prolonged in all infected groups. Trypanosoma rangeli seem to be less tolerant to higher temperatures since cultured-infected insects showed a reduction in the infection rates and passaged-infected insects decreased the salivary gland infection rates in those insects submitted to 30 °C. In vitro growth of T. rangeli was consistent with these results.

Trypanosoma cruzi-Trypanosoma rangeli co-infection ameliorates negative effects of single trypanosome infections in experimentally infected Rhodnius prolixus.

Trypanosoma cruzi, causative agent of Chagas disease, co-infects its triatomine vector with its sister species Trypanosoma rangeli, which shares 60% of its antigens with T. cruzi. Additionally, T. rangeli has been observed to be pathogenic in some of its vector species. Although T. cruzi-T. rangeli co-infections are common, their effect on the vector has rarely been investigated. Therefore, we measured the fitness (survival and reproduction) of triatomine species Rhodnius prolixus infected with just T. cruzi, just T. rangeli, or both T. cruzi and T. rangeli. We found that survival (as estimated by survival probability and hazard ratios) was significantly different between treatments, with the T. cruzi treatment group having lower survival than the co-infected treatment. Reproduction and total fitness estimates in the T. cruzi and T. rangeli treatments were significantly lower than in the co-infected and control groups. The T. cruzi and T. rangeli treatment group fitness estimates were not significantly different from each other. Additionally, co-infected insects appeared to tolerate higher doses of parasites than insects with single-species infections. Our results suggest that T. cruzi-T. rangeli co-infection could ameliorate negative effects of single infections of either parasite on R. prolixus and potentially help it to tolerate higher parasite doses.

Revisiting Trypanosoma rangeli Transmission Involving Susceptible and Non-Susceptible Hosts.

Trypanosoma rangeli infects several triatomine and mammal species in South America. Its transmission is known to occur when a healthy insect feeds on an infected mammal or when an infected insect bites a healthy mammal. In the present study we evaluated the classic way of T. rangeli transmission started by the bite of a single infected triatomine, as well as alternative ways of circulation of this parasite among invertebrate hosts. The number of metacyclic trypomastigotes eliminated from salivary glands during a blood meal was quantified for unfed and recently fed nymphs. The quantification showed that ~50,000 parasites can be liberated during a single blood meal. The transmission of T. rangeli from mice to R. prolixus was evaluated using infections started through the bite of a single infected nymph. The mice that served as the blood source for single infected nymphs showed a high percentage of infection and efficiently transmitted the infection to new insects. Parasites were recovered by xenodiagnosis in insects fed on mice with infections that lasted approximately four months. Hemolymphagy and co-feeding were tested to evaluate insect-insect T. rangeli transmission. T. rangeli was not transmitted during hemolymphagy. However, insects that had co-fed on mice with infected conspecifics exhibited infection rates of approximately 80%. Surprisingly, 16% of the recipient nymphs became infected when pigeons were used as hosts. Our results show that T. rangeli is efficiently transmitted between the evaluated hosts. Not only are the insect-mouse-insect transmission rates high, but parasites can also be transmitted between insects while co-feeding on a living host. We show for the first time that birds can be part of the T. rangeli transmission cycle as we proved that insect-insect transmission is feasible during a co-feeding on these hosts.

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.

Susceptibility of different Rhodnius species (Hemiptera, Reduviidae, Triatominae) to a Brazilian strain of Trypanosoma rangeli (SC58/KP1-).

INTRODUCTION: Specific host-parasite associations have been detected experimentally and suggest that triatomines of the genus Rhodnius act as biological filters in the transmission of Trypanosoma rangeli . OBJECTIVE: To analyze the susceptibility of four Rhodnius species ( Rhodnius robustus , Rhodnius neglectus , Rhodnius nasutus and Rhodnius pictipes ) to a Brazilian strain of T. rangeli (SC-58/KP1-). MATERIALS AND METHODS: We selected t hirty nymphs of each species, which were fed on blood infected with T. rangeli . Periodically, samples of feces and hemolymph were analyzed. Triatomines with T. rangeli in their hemolymph were fed on mice to check for transmission by bites. Later, the triatomines were dissected to confirm salivary gland infection. RESULTS: Specimens of R. pictipes showed higher rates of intestinal infection compared to the other three species. Epimastigotes and trypomastigotes were detected in hemolymph of four species; however, parasitism was lower in the species of the R. robustus lineage. Rhodnius robustus and R. neglectus specimens did not transmit T. rangeli by bite; after dissection, their glands were not infected. Only one specimen of R. nasutus and two of R. pictipes transmitted the parasite by bite. The rate of salivary gland infection was 16% for R. pictipes and 4% for R. nasutus . CONCLUSIONS: Both infectivity (intestinal, hemolymphatic and glandular) and transmission of T. rangeli (SC58/KP1-) were greater and more efficient in R. pictipes. These results reinforce the hypothesis that these triatomines may act as biological filters in the transmission of T. rangeli .

Rhodnius prolixus interaction with Trypanosoma rangeli: modulation of the immune system and microbiota population.

BACKGROUND: Trypanosoma rangeli is a protozoan that infects a variety of mammalian hosts, including humans. Its main insect vector is Rhodnius prolixus and is found in several Latin American countries. The R. prolixus vector competence depends on the T. rangeli strain and the molecular interactions, as well as the insect's immune responses in the gut and haemocoel. This work focuses on the modulation of the humoral immune responses of the midgut of R. prolixus infected with T. rangeli Macias strain, considering the influence of the parasite on the intestinal microbiota. METHODS: The population density of T. rangeli Macias strain was analysed in different R. prolixus midgut compartments in long and short-term experiments. Cultivable and non-cultivable midgut bacteria were investigated by colony forming unit (CFU) assays and by 454 pyrosequencing of the 16S rRNA gene, respectively. The modulation of R. prolixus immune responses was studied by analysis of the antimicrobial activity in vitro against different bacteria using turbidimetric tests, the abundance of mRNAs encoding antimicrobial peptides (AMPs) defensin (DefA, DefB, DefC), prolixicin (Prol) and lysozymes (LysA, LysB) by RT-PCR and analysis of the phenoloxidase (PO) activity. RESULTS: Our results showed that T. rangeli successfully colonized R. prolixus midgut altering the microbiota population and the immune responses as follows: 1 - reduced cultivable midgut bacteria; 2 - decreased the number of sequences of the Enterococcaceae but increased those of the Burkholderiaceae family; the families Nocardiaceae, Enterobacteriaceae and Mycobacteriaceae encountered in control and infected insects remained the same; 3 - enhanced midgut antibacterial activities against Serratia marcescens and Staphylococcus aureus; 4 - down-regulated LysB and Prol mRNA levels; altered DefB, DefC and LysA depending on the infection (short and long-term); 5 - decreased PO activity. CONCLUSION: Our findings suggest that T. rangeli Macias strain modulates R. prolixus immune system and modifies the natural microbiota composition.

Susceptibility of different Rhodnius species (Hemiptera, Reduviidae, Triatominae) to a Brazilian strain of Trypanosoma rangeli (SC58/KP1-) / Sensibilidad de diferentes especies de Rhodnius (Hemiptera, Reduviidae, Triatominae) a una cepa brasileña de Trypanosoma rangeli (SC58/KP1-)

Introduction: Specific host-parasite a ssociations have been detected experimentally and suggest that triatomines of the genus Rhodnius act as biological filters in the transmission of Trypanosoma rangeli . Objective: To analyze the susceptibility of four Rhodnius species ( Rhodnius robustus , Rhodnius neglectus , Rhodnius nasutus and Rhodnius pictipes ) to a Brazilian strain of T. rangeli (SC-58/KP1-). Materials and methods: We selected t hirty nymphs of each species, which were fed on blood infected with T. rangeli . Periodically, samples of feces and hemolymph were analyzed. Triatomines with T. rangeli in their hemolymph were fed on mice to check for transmission by bites. Later, the triatomines were dissected to confirm salivary gland infection. Results: Specimens of R. pictipes showed higher rates of intestinal infection compared to the other three species. Epimastigotes and trypomastigotes were detected in hemolymph of four species; however, parasitism was lower in the species of the R. robustus lineage. Rhodnius robustus and R. neglectus specimens did not transmit T. rangeli by bite; after dissection, their glands were not infected. Only one specimen of R. nasutus and two of R. pictipes transmitted the parasite by bite. The rate of salivary gland infection was 16% for R. pictipes and 4% for R. nasutus . Conclusions: Both infectivity (intestinal, hemolymphatic and glandular) and transmission of T. rangeli (SC58/KP1-) were greater and more efficient in R. pictipes. These results reinforce the hypothesis that these triatomines may act as biological filters in the transmission of T. rangeli .

Introducción. Se han detectado asociaciones biológicas huésped-parásito específicas que sugieren que los triatominos del género Rhodnius podrían actuar como filtros biológicos en la transmisión de Trypanosoma rangeli . Objetivo. Estudiar la sensibilidad de cuatro especies de Rhodnius ( Rhodnius robustus , Rhodnius neglectus , Rhodnius nasutus y Rhodnius p ictipes ) frente a la cepa de T. rangeli (SC-58/KP1-). Materiales y métodos. Se seleccionaron treinta ninfas de cada especie después de xenodiagnóstico artificial en sangre infectada con T. rangeli. Se examinaron periódicamente m uestras de heces y hemolinfa. Los insectos con hemolinfas infectadas fueron alimentados en ratones a fin de comprobar la transmisión por picadura y posteriormente disecados para confirmar la infección de las glándulas salivales . Resultados . En Rhodnius pictipes se encontró un mayor porcentaje de infección intestinal que en las otras especies . Se detectaron epimastigotes y tripomastigotes en la hemolinfa de las cuatro especies , y se encontró que el parasitismo fue menor en las especies del linaje R. robustus . Rhodnius robustus y R. neglectus no transmitían T. rangeli a ratones por picadura: después de la disección , sus glándulas no estaban infectadas. Solo un espécimen de R. nasutus y dos de R. pictipes transmitieron el parásito por la picadura . La tasa de infección glandular fue de 16 % para R. pictipes y de 4 % para R. nasutus . Conclusiones . La capacidad infecciosa ( hemolinfática, intestinal y glandular ) y la transmisión de T. rangeli (SC-58/KP1-) fueron mayores y más eficientes en R. pictipes . Estos resultados refuerzan la hipótesis de que estos triatominos actúan como filtros biológicos en la transmisión de T. rangeli .

Flight behavior and performance of Rhodnius pallescens (Hemiptera: Reduviidae) on a tethered flight mill.

ABSTRACT Flight dispersal of the triatomine bug species Rhodnius pallescens Barber, the principal vector of Chagas disease in Panama, is an important mechanism for spreading Trypanosoma cruzi, causative agent of Chagas disease. This study measures R. pallescens flight performance using a tethered flight mill both when uninfected, and when infected with T. cruzi or Trypanosoma rangeli. Forty-four out of the 48 (91.7%) insects initiated flight across all treatments, and trypanosome infection did not significantly impact flight initiation. Insects from all treatments flew a cumulative distance ranging from 0.5 to 5 km before fatiguing. The median cumulative distance flown before insect fatigue was higher in T. cruzi- and T. rangeli-infected insects than in control insects; however, this difference was not statistically significant. There was a positive relationship between parasite load ingested and time until flight initiation in T. rangeli-infected bugs, and T. rangeli- and T. cruzi-infected females flew significantly faster than males at different time points. These novel findings allow for a better understanding of R. pallescens dispersal ability and peridomestic management strategies for the prevention of Chagas disease in Panama.

Effects of infection by Trypanosoma cruzi and Trypanosoma rangeli on the reproductive performance of the vector Rhodnius prolixus.

The insect Rhodnius prolixus is responsible for the transmission of Trypanosoma cruzi, which is the etiological agent of Chagas disease in areas of Central and South America. Besides this, it can be infected by other trypanosomes such as Trypanosoma rangeli. The effects of these parasites on vectors are poorly understood and are often controversial so here we focussed on possible negative effects of these parasites on the reproductive performance of R. prolixus, specifically comparing infected and uninfected couples. While T. cruzi infection did not delay pre-oviposition time of infected couples at either temperature tested (25 and 30°C) it did, at 25°C, increase the e-value in the second reproductive cycle, as well as hatching rates. Meanwhile, at 30°C, T. cruzi infection decreased the e-value of insects during the first cycle and also the fertility of older insects. When couples were instead infected with T. rangeli, pre-oviposition time was delayed, while reductions in the e-value and hatching rate were observed in the second and third cycles. We conclude that both T. cruzi and T. rangeli can impair reproductive performance of R. prolixus, although for T. cruzi, this is dependent on rearing temperature and insect age. We discuss these reproductive costs in terms of potential consequences on triatomine behavior and survival.

Rhodnius prolixus: modulation of antioxidant defenses by Trypanosoma rangeli.

Trypanosoma rangeli is a protozoan parasite of insects and mammals that is challenged by the constant action of reactive oxygen species, generated either by its own metabolism or through the host immune response. The aim of this work was to investigate whether T. rangeli is able to modify the redox state of its insect vector, Rhodnius prolixus, through the modulation of such antioxidant enzymes as superoxide dismutase (SOD), catalase, and GPx present in the midgut of the insect. We verified that in R. prolixus fed with blood infected with T. rangeli there is an increase in SOD activity in the anterior and posterior midguts. However, the activities of enzymes related to hydrogen peroxide and hydroperoxides metabolism, such as catalase and GPx, were decreased in relation to the insect control group, which was only fed blood. These changes in the redox state of the vector led to an increase in lipid peroxidation and thiol oxidation levels in the anterior and posterior midgut tissues. We also verified that the addition of 1 mM GSH in the blood meal of the infected insects increased the proliferation of these parasites by 50%. These results suggest that there is an increase in oxidative stress in the insect gut during T. rangeli infection, and this condition could contribute to the control of the proliferation of these parasites.

The interaction between Trypanosoma rangeli and the nitrophorins in the salivary glands of the triatomine Rhodnius prolixus (Hemiptera; Reduviidae).

The parasite Trypanosoma rangeli develops in the intestinal tract of triatomines and, particularly in species of the genus Rhodnius, invades the hemolymph and salivary glands, where subsequent metacyclogenesis takes place. Many aspects of the interaction between T. rangeli and triatomines are still unclear, especially concerning the development of the parasite in the salivary glands and how the parasite interacts with the saliva. In this work, we describe new findings on the process of T. rangeli infection of the salivary glands and the impact of infection on the saliva composition. To ensure a complete infection (intestinal tract, hemolymph and salivary glands), 3rd instar Rhodnius prolixus nymphs were fed on blood containing T. rangeli epimastigotes using an artificial feeder. After molt to the 4th instar, the nymphs were inoculated with epimastigotes in the hemolymph. The results showed that the flagellates started to invade the salivary glands by the 7th day after the injection. The percentage of trypomastigotes inside the salivary glands continuously increased until the 25th day, at which time the trypomastigotes were more than 95% of the T. rangeli forms present. The salivary contents from T. rangeli-infected insects showed a pH that was significantly more acidic (<6.0) and had a lower total protein and hemeprotein contents compared with non-infected insects. However, the ratio of hemeprotein to total protein was similar in both control and infected insects. qPCR demonstrated that the expression levels of three housekeeping genes (18S rRNA, ß-actin and α-tubulin) and nitrophorins 1-4 were not altered in the salivary glands after an infection with T. rangeli. In addition, the four major nitrophorins (NPs 1-4) were knocked down using RNAi and their suppression impacted T. rangeli survival in the salivary glands to the point that the parasite burden inside the R. prolixus salivary glands was reduced by more than 3-fold. These results indicated that these parasites most likely non-specifically incorporated the proteins that were present in R. prolixus saliva as nutrients, without impairing the biosynthesis of the antihemostatic molecules.

Interaction between Trypanosoma rangeli and the Rhodnius prolixus salivary gland depends on the phosphotyrosine ecto-phosphatase activity of the parasite.

Trypanosoma rangeli is the trypanosomatid that colonizes the salivary gland of its insect vector, with a profound impact on the feeding capacity of the insect. In this study we investigated the role of the phosphotyrosine (P-Tyr) ecto-phosphatase activity of T. rangeli in its interaction with Rhodnius prolixus salivary glands. Long but not short epimastigotes adhered to the gland cells and the strength of interaction correlated with the enzyme activity levels in different strains. Differential interference contrast microscopy demonstrated that clusters of parasites are formed in most cases, suggesting cooperative interaction in the adhesion process. The tightness of the correlation was evidenced by modulating the P-Tyr ecto-phosphatase activity with various concentrations of inhibitors. Sodium orthovanadate, ammonium molybdate and zinc chloride decreased the interaction between T. rangeli and R. prolixus salivary glands in parallel. Levamisole, an inhibitor of alkaline phosphatases, affected neither process. EDTA strongly inhibited adhesion and P-Tyr ecto-phosphatase activity to the same extent, an effect that was no longer seen if the parasites were pre-incubated with the chelator and then washed. When the P-Tyr ecto-phosphatase of living T. rangeli epimastigotes was irreversibly inactivated with sodium orthovanadate and the parasite cells were then injected into the insect thorax, colonization of the salivary glands was greatly depressed for several days after blood feeding. Addition of P-Tyr ecto-phosphatase substrates such as p-nitrophenyl phosphate (pNPP) and P-Tyr inhibited the adhesion of T. rangeli to salivary glands, but P-Ser, P-Thr and ß-glycerophosphate were completely ineffective. Immunoassays using anti-P-Tyr-residues revealed a large number of P-Tyr-proteins in extracts of R. prolixus salivary glands, which could be potentially targeted by T. rangeli during adhesion. These results indicate that dephosphorylation of structural P-Tyr residues on the gland cell surfaces, mediated by a P-Tyr ecto-phosphatase of the parasite, is a key event in the interaction between T. rangeli and R. prolixus salivary glands.

Parasite-mediated interactions within the insect vector: Trypanosoma rangeli strategies.

Trypanosoma rangeli is a protozoan that is non-pathogenic for humans and other mammals but causes pathology in the genus Rhodnius. T. rangeli and R. prolixus is an excellent model for studying the parasite-vector interaction, but its cycle in invertebrates remains unclear. The vector becomes infected on ingesting blood containing parasites, which subsequently develop in the gut, hemolymph and salivary glands producing short and large epimastigotes and metacyclic trypomastigotes, which are the infective forms. The importance of the T. rangeli cycle is the flagellate penetration into the gut cells and invasion of the salivary glands. The establishment of the parasite depends on the alteration of some vector defense mechanisms. Herein, we present our understanding of T. rangeli infection on the vector physiology, including gut and salivary gland invasions, hemolymph reactions and behavior alteration.