Evaluation of the in vitro trypanocidal activity of triterpenes uvaol, betulinic acid and its semi-synthetic derivatives against the Y strain of Trypanosoma cruzi / Avaliação da atividade tripanocida in vitro dos triterpenos uvaol, ácido betulínico e seus derivados semissintéticos contra a cepa Y de Trypanosoma cruzi

Chagas disease is a neglected tropical disease that affects millions of people worldwide. Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas disease and its transmission occurs through blood meal by triatomine bugs, being oral transmission the most common form. More than 100 years after the disease´s discovery, benzonidazole is the only efficient drug against T. cruzi; however, this drug has numerous serious side effects and is only efficient in the acute phase of the disease. Natural products, such as triterpenes, have been an important source of new substances to combat human parasitology. In this study, two triterpenes, uvaol and betulinic acid, were tested against the parasite T. cruzi. The best results of in vitro tests were observed for uvaol with an IC50 value of 70.3 µM against the trypomastigost forms and an IC50 value of 90.6 µM against the amastigost forms. Three semi-synthetic derivatives of betulinic acid were obtained; the acetylated derivative showed excellent results against trypomastigotes forms (IC50 = 15.67 µM), but was not active against the amastigotes forms. The cytotoxic MTT test was also performed on LLCMK2 cells (Macaca mullata kidney epithelial cells) and betulinic acid showed the highest selectivity index (SI) with a value of 1.3.

Distribution and natural infection status of synantrophic triatomines (Hemiptera: Reduviidae), vectors of Trypanosoma cruzi, reveals new epidemiological scenarios for chagas disease in the Highlands of Colombia

Introduction Updating the distribution and natural infection status of triatomine bugs is critical for planning, prioritizing, and implementing strategies to control Chagas disease (CD), especially after vector reduction programs. After carrying out a control program, the Department of Boyaca contains the highest number of Colombian municipalities certified by PAHO to be free of intradomiciliary transmission of Trypanosoma cruzi by Rhodnius prolixus. The present work describes the spatial distribution, natural infection (NI), and molecular characterization of T. cruzi in synanthropic triatomines from the Department of Boyaca in 2017 and 2018. Materials and methods An entomological survey was conducted in 52 municipalities in Boyaca known to have had previous infestations of triatomine bugs. Insects were collected through active searches carried out by technical personnel from the Secretary of Health and community members using Triatomine Collection Stations (PITs-acronym in Spanish). For evaluation of natural infection, triatomines were identified morphologically and grouped in pools of one to five individuals of the same species collected in the same household. DNA derived from the feces of each pool of insects was analyzed by PCR for the presence of T. cruzi using primers flanking the satellite DNA of the parasite. SL-IR primers were used to differentiate TCI from the other DTUs and to identify different genotypes. The distribution of the collected triatomines was analyzed to determine any vector hotspots using spatial recreation. Results A total of 670 triatomine bugs was collected, belonging to five species: Triatoma dimidiata (73.2%), Triatoma venosa (16.7%), Panstrongylus geniculatus (5.7%), Rhodnius prolixus (4.4%), and Panstrongylus rufotuberculatus (0.4%), from 29 of the 52 municipalities. In total, 71.6% of the bugs were collected within houses (intradomiciliary) and the rest around the houses (peridomiciliary). Triatoma dimidiata was the most widely distributed species and had the highest natural infection index (37.8%), followed by T. venosa and P. geniculatus. TcI was the only DTU found, with the TcI Dom genotype identified in 80% of positive samples and TcI sylvatic in the other insects. Spatial analysis showed clusters of T. dimidiata and T. venosa in the northeast and southwest regions of Boyaca. Conclusions After some municipalities were certified free of natural transmission within houses (intradomiciliary transmission) of T. cruzi by R. prolixus, T. dimidiata has become the most prevalent vector present, and represents a significant risk of resurgent CD transmission. However, T. venosa, P. geniculatus, and P. rufotuberculatus also contribute to the increased risk of transmission. The presence of residual R. prolixus may undo the successes achieved through vector elimination programs. The molecular and spatial analysis used here allows us to identify areas with an ongoing threat of parasite transmission and improve entomological surveillance strategies.

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.

TriatoDex, an electronic identification key to the Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease: Development, description, and performance

Correct identification of triatomine bugs is crucial for Chagas disease surveillance, yet available taxonomic keys are outdated, incomplete, or both. Here we present TriatoDex, an Android app-based pictorial, annotated, polytomous key to the Triatominae. TriatoDex was developed using Android Studio and tested by 27 Brazilian users. Each user received a box with pinned, number-labeled, adult triatomines (33 species in total) and was asked to identify each bug to the species level. We used generalized linear mixed models (with user- and species-ID random effects) and information-theoretic model evaluation/averaging to investigate TriatoDex performance. TriatoDex encompasses 79 questions and 554 images of the 150 triatomine-bug species described worldwide up to 2017. TriatoDex-based identification was correct in 78.9% of 824 tasks. TriatoDex performed better in the hands of trained taxonomists (93.3% vs. 72.7% correct identifications; model-averaged, adjusted odds ratio 5.96, 95% confidence interval [CI] 3.09–11.48). In contrast, user age, gender, primary job (including academic research/teaching or disease surveillance), workplace (including universities, a reference laboratory for triatomine-bug taxonomy, or disease-surveillance units), and basic training (from high school to biology) all had negligible effects on TriatoDex performance. Our analyses also suggest that, as TriatoDex results accrue to cover more taxa, they may help pinpoint triatomine-bug species that are consistently harder (than average) to identify. In a pilot comparison with a standard, printed key (370 tasks by seven users), TriatoDex performed similarly (84.5% correct assignments, CI 68.9–94.0%), but identification was 32.8% (CI 24.7–40.1%) faster on average–for a mean absolute saving of ~2.3 minutes per bug-identification task. TriatoDex holds much promise as a handy, flexible, and reliable tool for triatomine-bug identification; an updated iOS/Android version is under development. We expect that, with continuous refinement derived from evolving knowledge and user feedback, TriatoDex will substantially help strengthen both entomological surveillance and research on Chagas disease vectors.

Neue Parameter für die Wirkstofftestung gegen Trypanosoma cruzi

Chagas disease is a zoonosis caused by Trypanosoma cruzi and transmitted by triatomine bugs. Autochthonous to Latin America, Chagas disease has spread globally through travel and migration. New drugs are needed urgently, in particular drugs that cure the chronic stage. This is where high-content imaging makes a key contribution: assays with fluorescent parasites in cell culture allow to determine pharmacodynamic parameters and to better assess the antichagasic potential of new molecules.

Extracellular Vesicles in Trypanosomatids: Host Cell Communication

Trypanosoma cruzi, Trypanosoma brucei and Leishmania (Trypanosomatidae: Kinetoplastida) are parasitic protozoan causing Chagas disease, African Trypanosomiasis and Leishmaniases worldwide. They are vector borne diseases transmitted by triatomine bugs, Tsetse fly, and sand flies, respectively. Those diseases cause enormous economic losses and morbidity affecting not only rural and poverty areas but are also spreading to urban areas. During the parasite-host interaction, those organisms release extracellular vesicles (EVs) that are crucial for the immunomodulatory events triggered by the parasites. EVs are involved in cell-cell communication and can act as important pro-inflammatory mediators. Therefore, interface between EVs and host immune responses are crucial for the immunopathological events that those diseases exhibit. Additionally, EVs from these organisms have a role in the invertebrate hosts digestive tracts prior to parasite transmission. This review summarizes the available data on how EVs from those medically important trypanosomatids affect their interaction with vertebrate and invertebrate hosts.

Preliminary Characterization of Triatomine Bug Blood Meals on the Island of Trinidad Reveals Opportunistic Feeding Behavior on Both Human and Animal Hosts

Chagas disease is a neglected tropical disease caused by infection with Trypanosoma cruzi. The parasite is endemic to the Americas, including the Caribbean, where it is vectored by triatomine bugs. Although Chagas disease is not considered a public health concern in the Caribbean islands, studies in Trinidad have found T. cruzi-seropositive humans and T. cruzi-infected triatomine bugs. However, little is known about triatomine bug host preferences in Trinidad, making it difficult to evaluate local risk of vector-borne T. cruzi transmission to humans. To investigate this question, we collected triatomine bugs in Trinidad and diagnosed each one for T. cruzi infection (microscopy and PCR). We then carried out a blood meal analysis using DNA extracted from each bug (PCR and sequencing). Fifty-five adult bugs (54 Panstrongylus geniculatus and one Rhodnius pictipes) were collected from five of 21 sample sites. All successful collection sites were residential. Forty-six out of the 55 bugs (83.6%) were infected with T. cruzi. Fifty-three blood meal hosts were successfully analyzed (one per bug), which consisted of wild birds (7% of all blood meals), wild mammals (17%), chickens (19%), and humans (57%). Of the 30 bugs with human blood meals, 26 (87%) were from bugs infected with T. cruzi. Although preliminary, our results align with previous work in which P. geniculatus in Trinidad had high levels of T. cruzi infection. Furthermore, our findings suggest that P. geniculatus moves between human and animal environments in Trinidad, feeding opportunistically on a wide range of species. Our findings highlight a critical need for further studies of Chagas disease in Trinidad in order to estimate the public health risk and implement necessary preventative and control measures.

The smORF-containing gene mille-pattes is required for moulting and Trypanosoma cruzi metacyclogenesis in the Chagas disease vector Rhodnius prolixus

SummaryChagas disease is estimated to affect 8 million people worldwide and is responsible for approximately 10,000 deaths in Latin America every year. Control of the triatomine bugs that transmit the flagellated parasite Trypanosoma cruzi has been the most successful strategy to avoid disease spread. Genes containing small open reading frames (smORFs, < 100 amino acids) constitute a putative reservoir of new vector control targets, since hundreds of these genes are present in insect genomes. Here, we show that the prototypic smORF-containing gene mille-pattes/polished-rice/tarsalless (mlpt/pri/tal) is essential for postembryonic development of the kissing bug Rhodnius prolixus and for T. cruzi metacyclogenesis during the nymphal stages. Injection of double-stranded RNA against mlpt (Rp-dsmlpt) during the nymphal stages leads to a plethora of phenotypes, which impair postembryonic development. First, fourth or fifth stage nymphs injected with Rp-dsmlpt do not moult even in the presence of the ecdysone receptor (EcR) mRNA. Second, Rp-dsmlpt nymphs have defects in gut morphology, delayed haemoglobin digestion, and decreased defecation volume compared with those of the control nymphs. Third, Rp-mlpt knockdown inhibits T. cruzi differentiation to the trypomastigote infective stage (metacyclogenesis) inside the R. prolixus gut. Overall, our study is the first to provide evidence that a smORF-containing gene regulates vector physiology and parasitic cycle thus enabling the development of novel molecular strategies to eliminate Chagas disease transmission.

Under pressure: phenotypic divergence and convergence associated with microhabitat adaptations in Triatominae

BackgroundTriatomine bugs, the vectors of Chagas disease, associate with vertebrate hosts in highly diverse ecotopes. When these blood-sucking bugs adapt to new microhabitats, their phenotypes may change. Although understanding phenotypic variation is key to the study of adaptive evolution and central to phenotype-based taxonomy, the drivers of phenotypic change and diversity in triatomines remain poorly understood.Methods/FindingsWe combined a detailed phenotypic appraisal (including morphology and morphometrics) with mitochondrial cytb and nuclear ITS2 DNA-sequence analyses to study Rhodnius ecuadoriensis populations from across the species’ range. We found three major, naked-eye phenotypic variants. Southern-Andean bugs (SW Ecuador/NW Peru) from house and vertebrate-nest microhabitats are typical, light-colored, small bugs with short heads/wings. Northern-Andean bugs (W Ecuador wet-forest palms) are dark, large bugs with long heads/wings. Finally, northern-lowland bugs (coastal Ecuador dry-forest palms) are light-colored and medium-sized. Wing and (size-free) head shapes are similar across Ecuadorian populations, regardless of habitat or naked-eye phenotype, but distinct in Peruvian bugs. Bayesian phylogenetic and multispecies-coalescent DNA-sequence analyses strongly suggest that Ecuadorian and Peruvian populations are two independently-evolving lineages, with little within-lineage structuring/differentiation.ConclusionsWe report sharp naked-eye phenotypic divergence of genetically similar Ecuadorian R. ecuadoriensis (house/nest southern-Andean vs. palm-dwelling northern bugs; and palm-dwelling Andean vs. lowland); and sharp naked-eye phenotypic similarity of typical, yet genetically distinct, southern-Andean bugs from house and nest (but not palm) microhabitats (SW Ecuador vs. NW Peru). This remarkable phenotypic diversity within a single nominal species likely stems from microhabitat adaptations possibly involving predator-driven selective pressure (yielding substrate-matching camouflage coloration) and a shift from palm-crown to vertebrate-nest microhabitats (yielding smaller bodies and shorter heads and wings). These findings shed new light on the origins of phenotypic diversity in triatomines, warn against excess reliance on phenotype-based triatomine-bug taxonomy, and confirm the Triatominae as an informative model-system for the study of phenotypic change under ecological pressure.Author summaryTriatomine bugs feed on the blood of vertebrates including humans and transmit the parasite that causes Chagas disease. The bugs, of which 150+ species are known, are highly diverse in size, shape, and color. Some species look so similar that they are commonly confused, whereas a few same-species populations look so different that they were thought to be separate species. Despite the crucial role of naked-eye phenotypes in triatomine-bug identification and classification (which are essential for vector control-surveillance), the origins of this variation remain unclear. Here, we describe a striking case of phenotypic divergence, with genetically similar bugs looking very different from one another, and phenotypic convergence, with bugs from two genetically distinct populations (likely on their way to speciation) looking very similar – and all within a single nominal species, Rhodnius ecuadoriensis. Phenotypically divergent populations occupy different ecological regions (wet vs. dry) and microhabitats (palm-crowns vs. vertebrate nests), whereas convergent populations occupy man-made and nest (but not palm) microhabitats. These findings suggest that triatomines can ‘respond’ to ecological novelty by changing their external, naked-eye phenotypes as they adapt to new microhabitats. We therefore warn that phenotypic traits such as overall size or color may confound triatomine-bug species identification and classification.