The Rpfor gene modulates the locomotory activity and host-seeking behaviour of Rhodnius prolixus.

The molecular bases of animal behaviour are intricate due to the pleiotropic nature of behaviour-modulating genes, which are often expressed across multiple tissues. The foraging gene (for) encodes a cGMP-dependent protein kinase (PKG), pivotal in regulating downstream target proteins through phosphorylation. In insects, for has been implicated in various behavioural contexts and physiological processes regarding searching for food. Rhodnius prolixus, a hematophagous bug that transmits Trypanosoma cruzi, the causative agent of Chagas disease, exhibits specific activity patterns associated with its hematophagous behaviour. Our previous work demonstrated a correlation between locomotor activity profiles and the expression of Rpfor, suggesting its involvement in modulating triatomine locomotion. In this study, we investigated the impact of Rpfor knockdown on locomotory activity, host-seeking behaviour, feeding performance and lipid metabolism in R. prolixus nymphs. Using RNA interference, we achieved a significant reduction of Rpfor expression in both the brain and fat body of R. prolixus nymphs. Knocked-down nymphs exhibited diminished non-oriented locomotory activity compared with controls, without altering the characteristic bimodal pattern of activity. Additionally, they displayed an increased tendency to approach a host, suggesting a role for Rpfor in modulating host-seeking behaviour. Feeding performance and lipid metabolism remained unaffected by Rpfor knockdown. Our findings underscore the multifaceted role of Rpfor in modulating locomotor activity and host-seeking behaviour in R. prolixus nymphs, shedding light on the molecular mechanisms underlying their hematophagous behaviour and potential implications for disease transmission. Further research is necessary to elucidate the intricate interplay between Rpfor expression, behaviour and physiological processes in triatomine bugs.

As bases moleculares do comportamento animal são complexas devido à natureza pleiotrópica dos genes envolvidos na sua modulação, normalmente expressos em múltiplos tecidos. O gene foraging (for) codifica para uma proteína quinase dependente de cGMP, fundamental para a regulação de proteínas alvo via fosforilação. Em insetos, o gene for tem sido associado a vários contextos comportamentais e processos fisiológicos relacionados com forrageamento. Rhodnius prolixus, um inseto hematófago que transmite Trypanosoma cruzi, o agente causativo da doença de Chagas, exibe padrões de atividade específicos associados com o seu comportamento hematófago. Em um estudo anterior, demonstramos uma correlação entre os perfis de atividade locomotora e a expressão de Rpfor, sugerindo o seu envolvimento na modulação da locomoção de triatomíneos. No presente estudo, investigamos o impacto do silenciamento de Rpfor na atividade locomotora, no comportamento de busca por hospedeiro, na performance alimentar, e no metabolismo de lipídeos em ninfas de R. prolixus. Através da técnica de RNA de interferência, obtivemos uma redução significativa da expressão do gene Rpfor no cérebro e no corpo gorduroso de R. prolixus. Insetos silenciados exibiram uma redução da atividade locomotora não orientada em comparação com controles, sem alterações no padrão bimodal da atividade. Adicionalmente, os insetos apresentaram um aumento no comportamento de busca por hospedeiro, sugerindo um papel para o Rpfor na sua modulação. A performance alimentar e o metabolismo de lipídeos não foram alterados pelo silenciamento do gene. Nossas descobertas ressaltam o papel multifuncional do gene Rpfor na modulação da atividade locomotora e no comportamento de busca por hospedeiro em R. prolixus, ampliando o conhecimento sobre os mecanismos moleculares relacionados ao seu comportamento hematófago e potenciais implicações para a transmissão de doenças. Estudos adicionais são necessários para elucidar a intrincada interação entre expressão, comportamento e processos fisiológicos de Rpfor em insetos triatomíneos.

The interplay between temperature, Trypanosoma cruzi parasite load, and nutrition: Their effects on the development and life-cycle of the Chagas disease vector Rhodnius prolixus.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi transmitted by blood-sucking insects of the subfamily Triatominae, is a major neglected tropical disease affecting 6 to 7 million of people worldwide. Rhodnius prolixus, one of the most important vectors of Chagas disease in Latin America, is known to be highly sensitive to environmental factors, including temperature. This study aimed to investigate the effects of different temperatures on R. prolixus development and life-cycle, its relationship with T. cruzi, and to gather information about the nutritional habits and energy consumption of R. prolixus. We exposed uninfected and infected R. prolixus to four different temperatures ranging from 24°C to 30°C, and monitored their survival, developmental rate, body and blood meal masses, urine production, and the temporal dynamics of parasite concentration in the excreted urine of the triatomines over the course of their development. Our results demonstrate that temperature significantly impacts R. prolixus development, life-cycle and their relationship with T. cruzi, as R. prolixus exposed to higher temperatures had a shorter developmental time and a higher mortality rate compared to those exposed to lower temperatures, as well as a lower ability to retain weight between blood meals. Infection also decreased the capacity of the triatomines to retain weight gained by blood-feeding to the next developmental stage, and this effect was proportional to parasite concentration in excreted urine. We also showed that T. cruzi multiplication varied depending on temperature, with the lowest temperature having the lowest parasite load. Our findings provide important insights into the potential impact of climate change on the epidemiology of Chagas disease, and can contribute to efforts to model the future distribution of this disease. Our study also raises new questions, highlighting the need for further research in order to understand the complex interactions between temperature, vector biology, and parasite transmission.

Effects of mating on female reproductive physiology in the insect model, Rhodnius prolixus, a vector of the causative parasite of Chagas disease.

The blood-sucking hemipteran Rhodnius prolixus is one of the main vectors of Chagas disease, a neglected tropical disease that affects several million people worldwide. Consuming a blood meal and mating are events with a high epidemiological impact since after each meal, mated females can lay fertile eggs that result in hundreds of offspring. Thus, a better knowledge of the control of R. prolixus reproductive capacity may provide targets for developing novel strategies to control vector populations, thereby reducing vector-host contacts and disease transmission. Here, we have used a combination of gene transcript expression analysis, biochemical assays, hormone measurements and studies of locomotory activity to investigate how mating influences egg development and egg laying rates in R. prolixus females. The results demonstrate that a blood meal increases egg production capacity and leads to earlier egg laying in mated females compared to virgins. Virgin females, however, have increased survival rate over mated females. Circulating juvenile hormone (JH) and ecdysteroid titers are increased in mated females, a process mainly driven through an upregulation of the transcripts for their biosynthetic enzymes in the corpus allatum and ovaries, respectively. Mated females display weaker locomotory activity compared to virgin females, mainly during the photophase. In essence, this study shows how reproductive output and behaviour are profoundly influenced by mating, highlighting molecular, biochemical, endocrine and behavioral features differentially expressed in mated and virgin R. prolixus females.

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.

Exposure to Trypanosoma parasites induces changes in the microbiome of the Chagas disease vector Rhodnius prolixus.

BACKGROUND: The causative agent of Chagas disease, Trypanosoma cruzi, and its nonpathogenic relative, Trypanosoma rangeli, are transmitted by haematophagous triatomines and undergo a crucial ontogenetic phase in the insect's intestine. In the process, the parasites interfere with the host immune system as well as the microbiome present in the digestive tract potentially establishing an environment advantageous for development. However, the coherent interactions between host, pathogen and microbiota have not yet been elucidated in detail. We applied a metagenome shotgun sequencing approach to study the alterations in the microbiota of Rhodnius prolixus, a major vector of Chagas disease, after exposure to T. cruzi and T. rangeli focusing also on the functional capacities present in the intestinal microbiome of the insect. RESULTS: The intestinal microbiota of R. prolixus was dominated by the bacterial orders Enterobacterales, Corynebacteriales, Lactobacillales, Clostridiales and Chlamydiales, whereas the latter conceivably originated from the blood used for pathogen exposure. The anterior and posterior midgut samples of the exposed insects showed a reduced overall number of organisms compared to the control group. However, we also found enriched bacterial groups after exposure to T. cruzi as well as T rangeli. While the relative abundance of Enterobacterales and Corynebacteriales decreased considerably, the Lactobacillales, mainly composed of the genus Enterococcus, developed as the most abundant taxonomic group. This applies in particular to vectors challenged with T. rangeli and at early timepoints after exposure to vectors challenged with T. cruzi. Furthermore, we were able to reconstruct four metagenome-assembled genomes from the intestinal samples and elucidate their unique metabolic functionalities within the triatomine microbiome, including the genome of a recently described insect symbiont, Candidatus Symbiopectobacterium, and the secondary metabolites producing bacteria Kocuria spp. CONCLUSIONS: Our results facilitate a deeper understanding of the processes that take place in the intestinal tract of triatomine vectors during colonisation by trypanosomal parasites and highlight the influential aspects of pathogen-microbiota interactions. In particular, the mostly unexplored metabolic capacities of the insect vector's microbiome are clearer, underlining its role in the transmission of Chagas disease. Video Abstract.

Metabolites as predictive biomarkers for Trypanosoma cruzi exposure in triatomine bugs.

Trypanosoma cruzi, the causative agent of Chagas disease (American trypanosomiasis), colonizes the intestinal tract of triatomines. Triatomine bugs act as vectors in the life cycle of the parasite and transmit infective parasite stages to animals and humans. Contact of the vector with T. cruzi alters its intestinal microbial composition, which may also affect the associated metabolic patterns of the insect. Earlier studies suggest that the complexity of the triatomine fecal metabolome may play a role in vector competence for different T. cruzi strains. Using high-resolution mass spectrometry and supervised machine learning, we aimed to detect differences in the intestinal metabolome of the triatomine Rhodnius prolixus and predict whether the insect had been exposed to T. cruzi or not based solely upon their metabolic profile. We were able to predict the exposure status of R. prolixus to T. cruzi with accuracies of 93.6%, 94.2% and 91.8% using logistic regression, a random forest classifier and a gradient boosting machine model, respectively. We extracted the most important features in producing the models and identified the major metabolites which assist in positive classification. This work highlights the complex interactions between triatomine vector and parasite including effects on the metabolic signature of the insect.

Triatoma infestans susceptibility to different Trypanosoma cruzi strains: parasite development and early escape from anterior midgut.

The escape kinetics from the anterior midgut (AM) of Trypanosoma cruzi during the initial steps of infection was assessed in Triatoma infestans, as well as its ability to survive migration in the digestive tract of the vector. All the four strains evaluated survived and reached variable parasite densities. After 49-50 days, YuYu [discrete typing units (DTU) I] strain reached the highest parasite numbers in the rectum followed by Bug (DTU V), CL-Brener (DTU VI) and Dm28c (DTU I). All strains accomplished metacyclogenesis. Bug strain reached the highest numbers of metacyclic trypomastigotes followed by YuYu and CL-Brener/Dm28c. A remarkable parasite reduction in the AM for Bug strain, but not Dm28c was noticed at 72 h of infection. In the posterior midgut + rectum high densities of parasites from both strains were detected at this period indicating the parasites crossed the AM. For Dm28c strain, in infections initiated with trypomastigotes, parasites left AM faster than those starting with epimastigotes. In conclusion, T. cruzi strains from different DTUs were able to infect T. infestans reaching variable parasite densities. The kinetics of migration in the digestive tract may be affected by strain and/or the evolutive form used for infection.

A Trypanosoma cruzi zinc finger protein that is implicated in the control of epimastigote-specific gene expression and metacyclogenesis.

Trypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programmed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and have their expression controlled by post-transcriptional mechanisms. Transcriptome analyses comparing three stages of the T. cruzi life cycle revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBPs), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP, named TcZH3H12, which contains a zinc finger domain and is up-regulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout (KO) epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Transcriptome analyses comparing wild type and TcZC3H12 KOs revealed a TcZC3H12-dependent expression of epimastigote-specific genes such as genes encoding amino acid transporters and proteins associated with differentiation (PADs). RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis.

Enzymatic biosynthesis of B-complex vitamins is supplied by diverse microbiota in the Rhodnius prolixus anterior midgut following Trypanosoma cruzi infection.

Trypanosoma cruzi, the causative agent of Chagas disease, colonizes the gut of triatomine insects, including Rhodnius prolixus. It is believed that this colonization upsets the microbiota that are normally present, presumably switching the environment to one more favorable for parasite survival. It was previously thought that one particular bacterium, Rhodococcus rhodnii, was essential for insect survival due to its ability to produce vital B-complex vitamins. However, these bacteria are not always identified in great abundance in studies on R. prolixus microbiota. Here we sequenced the microbiota of the insect anterior midgut using shotgun metagenomic sequencing in order to obtain a high-resolution snapshot of the microbes inside at two different time points and under two conditions; in the presence or absence of parasite and immediately following infection, or three days post-infection. We identify a total of 217 metagenomic bins, and recovered one metagenome-assembled genome, which we placed in the genus Dickeya. We show that, despite Rhodococcus being present, it is not the only microbe capable of synthesizing B-complex vitamins, with the genes required for biosynthesis present in a number of different microbes. This work helps to gain a new insight into the microbial ecology of R. prolixus.

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.

Modulation of IMD, Toll, and Jak/STAT Immune Pathways Genes in the Fat Body of Rhodnius prolixus During Trypanosoma rangeli Infection.

Trypanosoma rangeli is the second most common American trypanosome that infects man. It is vectored by triatomines from the genus Rhodnius, in which it invades the hemolymph and infects the salivary glands, avoiding the bug immune responses. In insects, these responses are initiated by well conserved pathways, mainly the IMD, Toll, and Jak/STAT. We hypothesize that long-term infection with T. rangeli in the gut or hemolymph of Rhodnius prolixus triggers different systemic immune responses, which influence the number of parasites that survive inside the vector. Thus, we investigated groups of insects with infections in the gut and/or hemolymph, and evaluated the parasite load and the expression in the fat body of transcription factors (Rp-Relish, Rp-Dorsal, and Rp-STAT) and inhibitors (Rp-Cactus and Rp-Caspar) of the IMD, Toll, and Jak/STAT pathways. We detected lower parasite counts in the gut of insects without hemolymph infection, compared to hemolymph-infected groups. Besides, we measured higher parasite numbers in the gut of bugs that were first inoculated with T. rangeli and then fed on infected mice, compared with control insects, indicating that hemolymph infection increases parasite numbers in the gut. Interestingly, we observed that genes from the three immune pathways where differentially modulated, depending on the region parasites were present, as we found (1) Rp-Relish downregulated in gut-and/or-hemolymph-infected insects, compared with controls; (2) Rp-Cactus upregulated in gut-infected insect, compared with controls and gut-and-hemolymph-infected groups; and (3) Rp-STAT downregulated in all groups of hemolymph-infected insects. Finally, we uncovered negative correlations between parasite loads in the gut and Rp-Relish and Rp-Cactus expression, and between parasite counts in the hemolymph and Rp-Relish levels, suggesting an association between parasite numbers and the IMD and Toll pathways. Overall, our findings reveal new players in R. prolixus-T. rangeli interactions that could be key for the capacity of the bug to transmit the pathogen.

Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut of Rhodnius prolixus but not in Triatoma infestans.

The protozoan Trypanosoma cruzi has the ability to spontaneously secrete extracellular vesicles (EVs). In this paper, T. cruzi EVs derived from epimastigote forms were evaluated during interaction with triatomine bugs Rhodnius prolixus and Triatoma infestans. T. cruzi EVs were purified and artificially offered to the insects prior to infection with epimastigote forms. No effect of EVs was detected in the parasite counts in the guts of both vectors after 49-50 days. On the other hand, pre-feeding with EVs delayed parasite migration to rectum only in the gut in R. prolixus after 21-22 days. Those data suggest a possible role of T. cruzi EVs during the earlier events of infection in the invertebrate host.

Effect of temperature and vector nutrition on the development and multiplication of Trypanosoma rangeli in Rhodnius prolixus.

Trypanosoma rangeli is a protozoan parasite that infects mammals and triatomines, causing different levels of pathogenicity in its invertebrate vectors, particularly those from the genus Rhodnius. We have recently shown that temperature can modulate T. rangeli growth during in vitro culture, as well as its in vivo pathogenicity to R. prolixus. In the present study, we investigated colonization of R. prolixus by T. rangeli and assessed the role of temperature and vector nutrition on parasite development and multiplication. We infected nymphs and either assessed parasite density in the first hours after the ingestion of the infected blood or maintained the nymphs for up to 60 days at different temperatures (21, 24, 27, and 30 °C) and under different blood-feeding schedules (either every 15 days, or on day 30 post infection only), with parasite development and multiplication measured on days 15, 30, and 60 post infection. In the first hours after ingesting infected blood, epimastigogenesis not only occurred in the anterior midgut, but a stable parasite population also established in this intestinal region. T. rangeli subsequently colonized all intestinal regions examined, but with fewer parasites being found in the rectum. The number of parasites was only affected by higher temperatures (27 and 30 °C) during the beginning of the infection (15 days post infection). Nutritional status of the vector also had a significant effect on parasite development, as reduced blood-feeding decreased infection rates by approximately 30%.

The molecular sensory machinery of a Chagas disease vector: expression changes through imaginal moult and sexually dimorphic features.

The triatomine bug Rhodnius prolixus is a main vector of Chagas disease, which affects several million people, mostly in Latin-America. Host searching, pheromone communication, and microclimatic preferences are aspects of its behaviour that depend on multimodal sensory inputs. The molecular bases of these sensory processes are largely unknown. The expression levels of genes transcribed in antennae were compared between 5th instar larvae, and female and male adults by means of RNA-Seq. The antennae of R. prolixus showed increased expression of several chemosensory-related genes in imaginal bugs, while both sexes had similar expression patterns for most target genes. Few cases suggest involvement of target genes in sexually dimorphic functions. Most odorant and ionotropic receptor genes seemed to be expressed in all libraries. OBPs and CSPs showed very high expression levels. Other sensory-related genes such as TRPs, PPKs and mechanoreceptors had consistent levels of expression in all libraries. Our study characterises most of the sensory gene repertoire of these insects, opening an avenue for functional genetics studies. The increase in expression of chemosensory genes suggests an enhanced role in adult bugs. This knowledge allows developing new behaviour interfering strategies, increasing the options for translational research in the vector control field.

Screening of Fungi for Biological Control of a Triatomine Vector of Chagas Disease: Temperature and Trypanosome Infection as Factors.

Entomopathogenic fungi have been investigated as an alternative tool for controlling various insects, including triatomine vectors of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Here we tested the pathogenicity and virulence of ten isolates of the fungi Metarhizium spp. and Beauveria bassiana against Rhodnius prolixus and found all of the isolates to be virulent. We used two isolates (URPE-11 Metarhizium anisopliae and ENT-1 Beauveria bassiana) for further screening based on their prolific sporulation in vitro (an important property of fungal biopesticides). We characterized their virulences in a dose-response experiment and then examined virulence across a range of temperatures (21, 23, 27 and 30°C). We found isolate ENT-1 to maintain higher levels of virulence over these temperatures than URPE-11. We therefore used B. bassiana ENT-1 in the final experiment in which we examined the survival of insects parasitized with T. cruzi and then infected with this fungus (once again over a range of temperatures). Contrary to our expectations, the survival of insects challenged with the pathogenic fungus was greater when they had previously been infected with the parasite T. cruzi than when they had not (independent of temperature). We discuss these results in terms of aspects of the biologies of the three organisms. In practical terms, we concluded that, while we have fungal isolates of potential interest for development as biopesticides against R. prolixus, we have identified what could be a critical problem for this biological tool: the parasite T. cruzi appears to confer a measure of resistance to the insect against the potential biopesticide agent so use of this fungus as a biopesticide could lead to selection for vector competence.

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.

A new antimicrobial protein from the anterior midgut of Triatoma infestans mediates Trypanosoma cruzi establishment by controlling the microbiota.

The Reduviid Triatoma infestans is a vector for the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. The parasite must address the defense molecules and microbiota that colonize the anterior midgut of T. infestans. To obtain insight into T. cruzi - microbiota interactions in triatomine insects, we characterized a new antimicrobial product from the anterior midgut of T. infestans (TiAP) that may be involved in these relationships. The TiAP DNA fragment was cloned and expressed in a bacterial system, and the effect of the protein on bacteria and T. cruzi was evaluated by RNAi, qPCR and antimicrobial experiments. The number of T. cruzi in T. infestans anterior midguts was significantly lower in TiAP knockdown insects than in unsilenced groups. We also verified that the amount of bacteria in silenced T. infestans is approximately 600-fold higher than in unsilenced insects by qPCR. The 327-bp cDNA fragment that encodes mature TiAP was cloned into the pET-14b vector and expressed fused to a His-tag in Escherichia coli C43. The recombinant protein (rTiAP) was purified using an Ni-NTA column, followed by a HiTrap SP column. According to a trypanocidal assay, rTiAP did not interfere with the viability of T. cruzi trypomastigotes. Moreover, in antimicrobial experiments using E. coli and Micrococcus luteus, the protein was only bacteriostatic for Gram-negative bacteria. The data indicate that infection by T. cruzi increases the expression of TiAP to modulate the microbiota. The inhibition of microbiota growth by TiAP is important for parasite establishment in the T. infestans anterior midgut.

A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus.

The triatomine insect, Rhodnius prolixus, is a vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. The parasite must overcome immune response and microbiota to develop inside the midgut of triatomines. In this study, we expressed, purified and characterized a Kazal-type inhibitor from the midgut of R. prolixus, named RpTI, which may be involved in microbiota - T. cruzi interactions. The qPCR showed that the RpTI transcript was primarily expressed in tissues from the intestinal tract and that it was upregulated in the anterior midgut after T. cruzi infection. A 315-bp cDNA fragment encoding the mature protein was cloned into the pPIC9 vector and expressed in Pichia pastoris system. Recombinant RpTI (rRpTI) was purified on a trypsin-Sepharose column and had a molecular mass of 11.5 kDa as determined by SDS-PAGE analysis. This protein inhibited trypsin (Ki = 0.42 nM), whereas serine proteases from the coagulation cascade were not inhibited. Moreover, trypanocidal assays revealed that rRpTI did not interfere in the viability of T. cruzi trypomastigotes. The RpTI transcript was also knocked down by RNA interference prior to infection of R. prolixus with T. cruzi. The amount of T. cruzi in the anterior midgut was significantly lower in RpTI knockdown insects compared to the non-silenced groups. We also verified that the bacterial load is higher in the anterior midgut of silenced and infected R. prolixus compared to non-silenced and infected insects. Our results suggest that T. cruzi infection increases the expression of RpTI to mediate microbiota modulation and is important for parasite immediately after infection with R. prolixus.

Trypanosoma cruzi, etiological agent of Chagas disease, is virulent to its triatomine vector Rhodnius prolixus in a temperature-dependent manner.

It is often assumed that parasites are not virulent to their vectors. Nevertheless, parasites commonly exploit their vectors (nutritionally for example) so these can be considered a form of host. Trypanosoma cruzi, a protozoan found in mammals and triatomine bugs in the Americas, is the etiological agent of Chagas disease that affects man and domestic animals. While it has long been considered avirulent to its vectors, a few reports have indicated that it can affect triatomine fecundity. We tested whether infection imposed a temperature-dependent cost on triatomine fitness. We held infected insects at four temperatures between 21 and 30°C and measured T. cruzi growth in vitro at the same temperatures in parallel. Trypanosoma cruzi infection caused a considerable delay in the time the insects took to moult (against a background effect of temperature accelerating moult irrespective of infection status). Trypanosoma cruzi also reduced the insects' survival, but only at the intermediate temperatures of 24 and 27°C (against a background of increased mortality with increasing temperatures). Meanwhile, in vitro growth of T. cruzi increased with temperature. Our results demonstrate virulence of a protozoan agent of human disease to its insect vector under these conditions. It is of particular note that parasite-induced mortality was greatest over the range of temperatures normally preferred by these insects, probably implying adaptation of the parasite to perform well at these temperatures. Therefore we propose that triggering this delay in moulting is adaptive for the parasites, as it will delay the next bloodmeal taken by the bug, thus allowing the parasites time to develop and reach the insect rectum in order to make transmission to a new vertebrate host possible.

Oxidative stress and DNA lesions: the role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability.

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.