Alessandra A. Guarneri

Characterization of Trypanosoma rangeli Strains Isolated in Central and South America: an Overview

Trypanosoma rangeli is a hemoflagelate parasite that infects domestic and sylvatic animals, as well as man, in Central and South America. T. rangeli has an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, sharing several animal reservoirs and triatomine vectors. We have isolated T. rangeli strains in the State of Santa Catarina, in southern Brazil, which dramatically increased the distribution area of this parasite. This brief review summarizes several studies comparing T. rangeli strains isolated in Santa Catarina with others isolated in Colombia, Honduras and Venezuela. The different methods used include indirect immunofluorescence and western blot assays, lectin agglutination, isoenzyme electrophoresis and random amplified polymorphic DNA analysis, triatomine susceptibility, in vitro cell infection assays, and mini-exon gene analysis.

Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR

BackgroundRhodnius prolixus is a blood-feeding insect that can transmit Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Recently, genomic resources for invertebrate vectors of human pathogens have increased significantly, and R. prolixus has been one of the main species studied among the triatomines. However, the paucity of information on many of the fundamental molecular aspects of this species limits the use of the available genomic information. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for the normalization of mRNA expression data from qPCR.ResultsThe expression stability of five candidate reference genes (18S rRNA, GAPDH, β-actin, α-tubulin and ribosomal protein L26) was evaluated by qPCR in two tissues (salivary gland and intestine) and under different physiological conditions: before and after blood feeding and after infection with T. cruzi or T. rangeli. The results were analyzed with three software programs: geNorm, NormFinder and BestKeeper. All of the evaluated candidate genes proved to be acceptable as reference genes, but some were found to be more appropriate depending on the experimental conditions. 18S, GAPDH and α-tubulin showed acceptable stability for studies in all of the tissues and experimental conditions evaluated. β-actin, one of the most widely used reference genes, was confirmed to be one of the most suitable reference genes in studies with salivary glands, but it had the lowest expression stability in the intestine after insect blood feeding. L26 was identified as the poorest reference gene in the studies performed.ConclusionsThe expression stability of the genes varies in different tissue samples and under different experimental conditions. The results provided by three statistical packages emphasize the suitability of all five of the tested reference genes in both the crop and the salivary glands with a few exceptions. The results emphasise the importance of validating reference genes for qRT-PCR analysis in R. prolixus studies.

Influence of the Blood Meal Source on the Development of Triatoma infestans, Triatoma brasiliensis, Triatoma sordida, and Triatoma pseudomaculata (Heteroptera, Reduviidae)

Abstract The objective of this study was to determine the influence of the blood meal source on the life cycle and reproductive development of female Triatoma infestans (Klug, 1834), Triatoma brasiliensis Neiva, 1911, Triatoma sordida (Stal, 1859), and Triatoma pseudomaculata Corrêa & Espínola, 1964. In all triatomine species studied the life cycle was shorter for the groups fed on mice than for those fed on pigeons, the range of differences being between 1.5 times (T. pseudomaculata and T. infestans) and 2.4 times (T. brasiliensis). The mortality rate of nymphs during the life cycle tended to be greater in insects fed on pigeons than in those fed on mice, the differences for T. brasiliensis being statistically significant. Females of T. sordida and T. pseudomaculata had a greater fecundity than those of T. infestans and T. brasiliensis independently of the blood meal source. The differences of fecundity observed probably reflect differences in the availability of blood in the silvatic ecotopes of these species, meals being more frequent for T. infestans and T. brasiliensis, which live at high densities in association with rodents in highly stable ecotopes. Because T. sordida and T. pseudomaculata live in more unstable ecotopes with fewer sources of blood they form small sparse colonies and invest more energy in reproduction than maintenance.

Different serological cross-reactivity of Trypanosoma rangeli forms in Trypanosoma cruzi-infected patients sera

BackgroundAmerican Trypanosomiasis or Chagas disease is caused by Trypanosoma cruzi which currently infects approximately 16 million people in the Americas causing high morbidity and mortality. Diagnosis of American trypanosomiasis relies on serology, primarily using indirect immunofluorescence assay (IFA) with T. cruzi epimastigote forms. The closely related but nonpathogenic Trypanosoma rangeli has a sympatric distribution with T. cruzi and is carried by the same vectors. As a result false positives are frequently generated. This confounding factor leads to increased diagnostic test costs and where false positives are not caught, endangers human health due to the toxicity of the drugs used to treat Chagas disease.ResultsIn the present study, serologic cross-reactivity between the two species was compared for the currently used epimastigote form and the more pathologically relevant trypomastigote form, using IFA and immunoblotting (IB) assays. Our results reveal an important decrease in cross reactivity when T. rangeli culture-derived trypomastigotes are used in IFA based diagnosis of Chagas disease. Western blot results using sera from both acute and chronic chagasic patients presenting with cardiac, indeterminate or digestive disease revealed similar, but not identical, antigenic profiles.ConclusionThis is the first study addressing the serological cross-reactivity between distinct forms and strains of T. rangeli and T. cruzi using sera from distinct phases of the Chagasic infection. Several T. rangeli-specific proteins were detected, which may have potential as diagnostic tools.

A standardizable protocol for infection of Rhodnius prolixus with Trypanosoma rangeli, which mimics natural infections and reveals physiological effects of infection upon the insect

Trypanosoma rangeli is a protozoan parasite that shares hosts - mammals and triatomines - with Trypanosoma cruzi, the etiological agent of Chagas disease. Although T. rangeli is customarily considered to be non-pathogenic to human hosts, it is able to produce pathologies in its invertebrate hosts. However, advances are hindered by a lack of standardization of infection procedures and these pathologies need documentation. To establish a suitable, and standardizable, infection protocol, the duration of the fourth instar was evaluated in nymphs infected by injection into the thorax with different concentrations of parasites, and compared with nymphs infected naturally (i.e. orally). We demonstrate that delays in moult were attributable to the presence of the parasite in the haemolymph (vs. the gut) and propose that the protocol presented here simulates closely natural infections. This methodology was then used for the evaluation of physiological parameters and several hitherto unreported effects of T. rangeli infection on Rhodnius prolixus were revealed. Haemolymph volume was greater in infected than uninfected nymphs but this alteration could not be attributed to water retention, since infected insects lost the same amount of water as controls. However, we found that lipid content and fat body weight were both increased in insects infected by T. rangeli. We propose that this is due to the parasites sequestration of host blood lipids and carrier proteins. With these findings, we have taken a few first steps to unravelling physiological details of the host-parasite interaction. We suggest future directions towards a fuller understanding of mechanistic and adaptive aspects of triatomine-trypanosomatid interactions.

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.

Electromyogram of the Cibarial Pump and the Feeding Process in Hematophagous Hemiptera

The feeding process of hematophagous insects constitutes an important period in their life cycle. During this process, the insect is in close contact with its host, the blood required for reproduction and completion of insect development is obtained and pathogens can be transmitted. Several studies have focused on this period using different tools to monitor the details of feeding, with particular attention given to electromyograms of the cibarial pump. The cibarial pump is a structure associated with a complex of muscles located in the head of the insect. It controls the ingestion of blood from the host and transfers it to the gut. Monitoring of the frequency and shape of the electrical signals produced by this muscle complex enables the phases and parameters of the feeding process to be precisely determined and allows any disturbance or feature that may affect hematophagy to be evaluated. In this chapter we review technical and methodological issues that allow cibarial pump electromyograms to be used in studying the insect blood-feeding process and describe the results that may be obtained from such a system.

Population Biology of Rhodnius domesticus Neiva & Pinto, 1923 (Hemiptera: Reduviidae) under Laboratory Conditions

The entire life cycle of Rhodnius domesticus, fed weekly on mice, was studied under controlled conditions. Aspects related to hatching, life time, mortality, feeding behaviour and fecundity for each stage of the insect life-cycle were evaluated. The hatching rate observed in 100 eggs was 57% and the mean time of hatching was 15.6 days. Forty-six nymphs (80.7%) completed the cycle and the mean time from NI to adult was 93.8 days. The average span in days for each stage was 12.4 for NI, 9.8 for NII, 14.2 for NIII, 16.8 for NIV and 25.0 NV. the number of bloodmeals in each nymphal stage varied from 1 to 3. The mortality rate was 12.3% for NI, 3.5% for NII and 1.7% for NIII and NV nymphs. The mean number of eggs laid per female in a 9-month period was 333.1. Average adult survival rates were 287.6 + 133 and 328 + 73 days for males and females respectively.

Relative humidity and water loss in Triatoma brasiliensis

Abstract.  The effect of the relative humidity (RH) of the environment on water loss is studied in Triatoma brasiliensis. Starved or recently fed early stages of T. brasiliensis are exposed to 15, 37, 46 and 73% RH. Water loss is estimated by weighing individual insects daily. The results obtained with starved insects show that the group maintained at 15% RH loses more water and shows a higher mortality compared with the others. An experiment in which recently fed nymphs are exposed to different RHs shows that all groups of insects lose the greatest amount of water during the first day. The group exposed to 15% RH show a higher water loss than the others before and after ecdysis. The survival of recently fed nymphs is not affected by the level of RH to which they are exposed. The results are discussed in relation to the water balance in this species.

Feeding Performance of Triatoma brasiliensis (Hemiptera: Reduviidae) on Habitual Hosts: Thrichomys laurentius (Rodentia: Echimyidae) and Humans

Triatoma brasiliensis is an important vector of Trypanosoma cruzi in the semiarid zones of Northeastern Brazil where it is commonly found in human dwellings, peridomiciliary areas, and the wild environments. To better understand features that may be influencing the interaction of T. brasiliensis with its hosts, in the present work we compared the feeding performance of sylvatic and domestic populations of T. brasiliensis on humans and on Thrichomys laurentius, a wild rodent found in the natural ecotopes of T. brasiliensis. The analyses were performed using the electronic monitoring of the cibarial pump. The blood sources considerably affected the feeding parameters. Insects fed on T. laurentius had lower ingestion rate, gained less weight, had longer interruption time, remained less time in contact with the host, and had the contractions of the pump in a lower frequency during the feeding process in comparison to insects fed on humans. These results indicate that humans are better blood sources for T. brasiliensis than T. laurentius. The fact that there was no difference between the feeding performance of sylvatic and domestic insects suggests that feeding is not a barrier for the domiciliation. Together, these findings suggest that shelter and faster blood meals are qualities of human dwellings that facilitate the domiciliation of T. brasiliensis.