Lessons to be popular: the chemical basis of aggregation in Trypanosoma cruzi-infected and non-infected Chagasic bugs.

Aggregation is one of the most remarkable behaviours in the animal kingdom-a process that is usually governed by pheromones. Triatomines are blood-sucking bugs that act as vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in mammals, including humans. Triatomines usually gather in roosting refuges by using aggregation pheromones of unknown chemical structure. In terms of vector control, one option to reduce triatomine-human contact is via capturing the insects into traps baited with lures based on such aggregation pheromones. As a first step towards this aim, we elucidated the aggregation pheromone in the triatomine Triatoma pallidipennis, using T. cruzi-infected and non-infected bugs. We used different extraction techniques and gas chromatography coupled to mass spectrometry for the identification. Also, two different bioassays were implemented for evaluating the attractant and arrestant activity of the pheromone. We found that T. pallidipennis produced short-chain aldehydes as attractants, and nitrogen-derived compounds as arrestants. We detected differences in the production and perception of these compounds according to whether animals were infected or not. These findings show that T. cruzi may influence triatomine chemical ecology and are promising tools for triatomine control.

Identification of Discrete Typing Units of Trypanosoma cruzi Isolated from Domestic Environments in Southeastern Mexico.

Background: The Trypanosoma cruzi parasite is the causal agent of Chagas disease, recognized by the World Health Organization as a neglected tropical disease. Currently there are seven discrete typing units (DTUs) of T. cruzi distributed in America, but there are still gaps about its distribution in some endemic regions. Materials and Methods: Seventeen units isolated from Chiapas and Oaxaca in Mexico were identified by amplification of the C-5 sterol desaturase gene. Results: Three DTUs of T. cruzi, TcI (6), TcII (10), and TcIV (1) were detected by comparing polymorphic sites in specific regions. Conclusions: New DTUs are reported for both states, where TcII was the most common DTU. The genetic characterization of the isolates can help to understand the epidemiology of Chagas disease.

Acute toxicity of two insecticides on two species of Chagas disease vectors.

The control of triatomine vectors of Chagas disease is mainly based on the use of pyrethroid insecticides. Because chemical control is the primary method for managing these insects, it is crucial to diversify the range of products utilized to mitigate the risk of resistance development. This study evaluated the toxicity of two insecticides with different modes of action on Triatoma dimidiata Latreille and T. pallidipennis Stal first and third instar nymphs. Our study focused on the effects of two insecticides, buprofezin (a growth regulator) and flunocamid (an anti-feeder), on the mortality rate of triatomine bugs in a laboratory setting. Moreover, we investigated how direct and indirect (film method) exposure to these insecticides impacted the survival of the insects. Flonicamid emerged as a promising insecticide for triatomine control since it caused 100% mortality in first-instar nymphs 48 h after direct exposure. While, in third instar nymphs, the maximum mortality was 88% at 72 h after exposure. Our result can be used as a basis for future triatomine control plans.

Body shape and fluctuating asymmetry following different feeding sources and feeding time in a triatomine, Triatoma pallidipennis (Stål, 1892).

Even when an animal has a generalist diet, different food sources can impact its body shape and fluctuating asymmetry (a stress indicator; FA). To test this, we varied the food source (mammalian, avian or defibrinated mammalian blood; and control animals - ad libitum feeding) and the time of feeding (every 8 days, 45 days and ad libitum) having the Chagas triatomine vector, Triatoma pallidipennis (Stål, 1892), as a study animal which has presumable generalist feeding habits. This factorial design was applied since first instar animals until adulthood. As response variables, we measured body shape and FA in adults of both sexes, using a two-dimensional geometric morphometrics protocol. The highest variance in body shape was explained by diet (17%), followed by sex nested within diet (12%). Males had less morphological differentiation than females: females with defibrinated blood provided every 45 days differentiated more, while those that fed on mammalian blood every 8 days differed less. Distances among the averages of the FA component related to shape indicated greater distances between avian blood provided every 45 days and mammalian blood provided every 8 days, as well as between the two groups fed on avian blood (feeding every 8 and 45 days), followed by avian and defibrinated blood, both fed every 8 days. These results indicate that blood source and feeding time have significant effects on the body shape, and FA in females and both sexes. Thus, despite general feeding habits, avian blood showed a greater impact on shape and FA in triatomines. This may select for triatomines to use mammal blood rather than avian blood if they have the chance to do so.

Higher temperatures reduce the number of Trypanosoma cruzi parasites in the vector Triatoma pallidipennis.

BACKGROUND: Relatively little is known about how pathogens transmitted by vector insects are affected by changing temperatures analogous to those occurring in the present global warming scenario. One expectation is that, like their ectothermic vectors, an increase in temperature could reduce their fitness. Here, we have investigated the effect of high temperatures on the abundance of Trypanosoma cruzi parasites during infection in the vector Triatoma pallidipennis. METHODS: We exposed T. pallidipennis nymphs to two strains (Morelos and Chilpancingo) of T. cruzi. Once infected, the fifth-instar bugs were distributed among three different temperature groups, i.e. 20, 30, and 34 °C, and the resulting parasites were counted when the bugs reached adulthood. RESULTS: The number of parasites increased linearly with time at 20 °C and, to a lesser extent, at 30 °C, especially in the Chilpancingo compared to the Morelos strain. Conversely, at 34 °C, the number of parasites of both strains decreased significantly compared to the other two temperatures. CONCLUSIONS: These results suggest negative effects on the abundance of T. cruzi in T. pallidipennis at high temperatures. This is the first evidence of the effect of high temperatures on a pathogenic agent transmitted by an insect vector in the context of global warming. Further tests should be done to determine whether this pattern occurs with other triatomine species and T. cruzi strains.

Zombie bugs? Manipulation of kissing bug behavior by the parasite Trypanosoma cruzi.

The parasite manipulation hypothesis states that the parasite modifies host's behavior thereby increasing the probability that the parasite will pass from an intermediate host to its final host. We used the kissing bugs Triatoma pallidipennis and T. longipennis and two isolates of the Trypanosoma cruzi parasite (Chilpancingo and Morelos) to test these ideas. These insects are intermediate hosts of this parasite, which is the causal agent of Chagas disease. The Chilpancingo isolate is more pathogenic than the Morelos isolate, in the bugs. We expected that infected bugs would be more active and likely at detecting human-like odors. Given the differences in pathogenicity between isolates, we expected the Chilpancingo isolate to induce these effects more strongly and lead to higher parasite number than the Morelos isolate. Finally, infected bugs would gain less mass (a mechanism thought to increase bite rate, and thus transmission) than non-infected bugs. Having determined that both isolate haplotypes belong to the Tc1a group, we found that: (a) young instars of both species were more active and likely to detect human odor when they were infected, regardless of the isolate; (b) there was no difference in parasite abundance depending on isolate; and, (c) infected bugs did not end up with less weight than uninfected bugs. These results suggest that T. cruzi can manipulate the bugs, which implies a higher risk to contract Chagas disease than previously thought.

The cost of being a killer's accomplice: Trypanosoma cruzi impairs the fitness of kissing bugs.

Relatively little is known about the fitness effects and life history trade-offs in medically important parasites and their insect vectors. One such case is the triatomine bugs and the parasite Trypanosoma cruzi, the key actors in Chagas disease. Previous studies have revealed some costs but have not simultaneously examined traits related to development, reproduction, and survival or their possible trade-offs. In addition, these studies have not compared the effects of genetically different T. cruzi strains that differ in their weakening effects in their vertebrate hosts. We compared the body size of the bugs after infection, the number of eggs laid, hatching/non-hatching rate, hatching success, survival, and the resulting number of parasites in Meccus (Triatoma) pallidipennis bugs that were experimentally infected with two strains of T. cruzi (Chilpancingo [CH], the most debilitating in vertebrates; and Morelos [MO], the least debilitating) (both belonging to TcI group). Our results showed that infection affects size (MO < CH; MO and CH = control), number of eggs laid (MO and CH < control) hatching/non-hatching rate (MO < control < CH), hatching success (control < MO, CH = control = MO), and survival (Chilpancingo < Morelos < control). In addition, the CH strain produced more parasites than the MO strain. These results suggest that (a) infection costs depend on the parasite's origin, (b) the more debilitating effects of the CH strain are due to its increased proliferation in the host, and (c) differences in pathogenicity among T. cruzi strains can be maintained through their different effects on hosts' life history traits. Probably, the vectorial capacity mediated by a more aggressive strain could be reduced due to its costs on the triatomine, leading to a lower risk of vertebrate and invertebrate infection in natural populations.

A reduction in ecological niche for Trypanosoma cruzi-infected triatomine bugs.

BACKGROUND: Theory predicts that parasites can affect and thus drive their hosts' niche. Testing this prediction is key, especially for vector-borne diseases including Chagas disease. Here, we examined the niche use of seven triatomine species that occur in Mexico, based on whether they are infected or not with Trypanosoma cruzi, the vectors and causative parasites of Chagas disease, respectively. Presence data for seven species of triatomines (Triatoma barberi, T. dimidiata, T. longipennis, T. mazzottii, T. pallidipennis, T. phyllosoma and T. picturata) were used and divided into populations infected and not infected by T. cruzi. Species distribution models were generated with Maxent 3.3.3k. Using distribution models, niche analysis tests of amplitude and distance to centroids were carried out for infected vs non-infected populations within species. RESULTS: Infected populations of bugs of six out of the seven triatomine species showed a reduced ecological space compared to non-infected populations. In all but one case (T. pallidipennis), the niche used by infected populations was close to the niche centroid of its insect host. CONCLUSIONS: Trypanosoma cruzi may have selected for a restricted niche amplitude in triatomines, although we are unaware of the underlying reasons. Possibly the fact that T. cruzi infection bears a fitness cost for triatomines is what narrows the niche breadth of the insects. Our results imply that Chagas control programmes should consider whether bugs are infected in models of triatomine distribution.

Trypanosoma cruzi: A review of biological and methodological factors in Mexican strains.

Trypanosoma cruzi, responsible for Chagas disease, is a serious public health problem in Latin America with eight million people infected in the world. Clinical manifestations observed in humans due to T. cruzi infection are largely associated with the wide biological and genetic heterogeneity of the parasite. This review presents an overview of the parasitological aspects of various strains of T. cruzi isolated mainly in Mexico, as well as an analysis of the methodological processes used to determine their virulence that could be influencing their biological characterization. We emphasize the importance of using uniform protocols to study T. cruzi virulence, taking into account factors related to: strain (i.e. developmental stage, lineage, biological origin, genetic variability), animal model used (i.e. role of hormones, host immune response, age) and methodology (i.e. inoculum size, inoculation route, and laboratory conditions used during strain maintenance). These uniform protocols will then allow proposing elements for understanding clinical evolution and management of the disease, for providing adequate treatment, and for developing tools for future vaccines against Chagas disease.

Glycosylation on proteins of the intestine and perimicrovillar membrane of Triatoma (Meccus) pallidipennis, under different feeding conditions.

Trypanosoma cruzi, the causative agent of Chagas disease, interacts with molecules in the midgut of its insect vector to multiply and reach the infective stage. Many studies suggest that the parasite binds to midgut-specific glycans. We identified several glycoproteins expressed in the intestine and perimicrovillar membrane (PMM) of Triatoma (Meccus) pallidipennis under different feeding conditions. In order to assess changes in protein-linked glycans, we performed lectin and immunoblot analyses on glycoprotein extracts from these intestinal tissues using well-characterized lectins, and an antibody, which collectively recognize a wide range of different glycans epitopes. We observed that the amount and composition of proteins and glycoproteins associated with different glycans structures changed over time in the intestines and PMM under different physiological conditions. PMM extracts contained a wide variety of glycoproteins with different sugar residues, including abundant high-mannose and complex sialylated glycans. We propose that these molecules could be involved in the process of parasite-vector interactions.

Effects of Trypanosoma cruzi on the phenoloxidase and prophenoloxidase activity in the vector Meccus pallidipennis (Hemiptera: Reduviidae).

BACKGROUND: Triatomine insects are vectors of Trypanosoma cruzi, the causal agent of Chagas disease. The insect-parasite interaction has been studied in relation to the transmission and prevalence of this disease. For most triatomines, however, several crucial aspects of the insect immune response are still unknown. For example, only for Rhodnius prolixus and Triatoma infestans has the activity of phenoloxidase (PO) and its zymogen prophenoloxidase (proPO) been reported in relation to the hemolymph and anterior midgut (AM). The aim of this study was to gain insight into the immune response to T. cruzi infection of an important triatomine in Mexico, Meccus pallidipennis. METHODS: Parasites were quantified in the rectal contents of infected M. pallidipennis groups. We examined some key factors in disease transmission, including the systemic (hemolymph) and local (gut) immune response. RESULTS: Parasites were present in the rectal contents at 4 days post-infection (pi) and reached their maximum density on day 7 pi. At 7 and 9 days pi mainly metacyclic trypomastigotes occurred. Compared to the control, the infected insects exhibited diminished PO activity in the hemolymph on days 9, 16 and 20 pi, and in the AM only on day 9. Additionally, infected insects displayed lower proPO activity in the hemolymph on day 1, but greater activity in the AM on day 28. CONCLUSIONS: The parasite strain originating from M. pallidipennis rapidly colonized the rectum of nymphs of this triatomine and developed high numbers of metacyclic trypomastigotes. Neither the changes of concentrations of PO and proPO in the hemolymph nor in the AM correlated with the changes in the population of T. cruzi.

What makes an effective Chagas disease vector? Factors underlying Trypanosoma cruzi-triatomine interactions.

The Chagas disease is caused by the parasite Trypanosoma cruzi, which infect blood-feeding triatomine bugs to finally reach mammal hosts. Chagas disease is endemic in Latin America, and is ranked among the 13 neglected tropical diseases worldwide. Currently, an estimate of 7 million people is infected by T. cruzi, leading to about 22 000 deaths per year throughout the Americas. As occurs with other vectors, a major question towards control programs is what makes a susceptible bug. In this review, we focus on findings linked to insect gut structure and microbiota, immunity, genetics, blood sources, abiotic factors (with special reference to ambient temperature and altitude) to understand the interactions occurring between T. cruzi and triatomine bugs, under a co-evolutionary scenario. These factors lead to varying fitness benefits and costs for bugs, explaining why infection in the insect takes place and how it varies in time and space. Our analysis highlights that major factors are gut components and microbiota, blood sources and temperature. Although their close interaction has never been clarified, knowledge reviewed here may help to boost the success of triatomine control programs, reducing the use of insecticides.

Possible Differences in the Effects of Trypanosoma cruzi on Blood Cells and Serum Protein of Two Wildlife Reservoirs.

A key step in the dynamics of vector-borne diseases is the role of seasonality. Trypanosoma cruzi is a protozoan that causes Chagas disease. Some wild mammals are considered natural hosts, yet not all mammals show the same response to infection. We explored the effect of T. cruzi on blood parameters in two mammal carnivores, coati (Nasua narica) and raccoon (Procyon lotor), that were naturally infected in summer and winter seasons. The study was carried out in the Zoological Park "Parque Museo de la Venta," in Southeastern Mexico. Blood samples were collected in summer and winter from 2010 to 2013. Parasite infection was assessed by PCR from whole blood, and a complete hemogram was determined by traditional manual methods. We found that both species had the same T. cruzi I lineage. For coatis, mean corpuscular volume, mean corpuscular hemoglobin, and monocytes were dependent of season, while eosinophils and plasma proteins were significantly different, but with no season effect. For raccoon, erythrocytes, mean corpuscular volume, mean corpuscular hemoglobin, and monocytes were dependent of season. These results and a previous study that indicated interspecific differences in parasitemia in both species suggest that raccoon is a better reservoir than coati. Such a different interspecific response implies that animals do not contribute equally to maintain T. cruzi parasites in the ecosystem. Such inequality differs according to season.

Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae).

Hemipterans and thysanopterans (Paneoptera: Condylognatha) differ from other insects by having an intestinal perimicrovillar membrane (PMM) which extends from the base of the microvilli to the intestinal lumen. The development and composition of the PMM in hematophagous Reduviidae depend on factors related to diet. The PMM may also allow the human parasite Trypanosoma cruzi, the etiological agent of human Chagas Disease, to establish and develop in this insect vector. We studied the PMM development in the Mexican vector of Chagas Disease, Triatoma (Meccus) pallidipennis. We describe changes in the midgut epithelial cells of insects in response to starvation, and at different times (10, 15 and 20 days) after bloodfeeding. In starved insects, the midguts showed epithelial cells closely connected to each other but apparently free of PMM with some regions being periodic acid-Schiff (PAS-Schiff) positive. In contrast, the PMM was evident and fully developed in the midgut region of insects 15 days after feeding. After this time, the PMM completely covered the microvilli and reached the midgut lumen. At 15 days following feeding the labeled PAS-Schiff increased in the epithelial apex, suggesting an increase in carbohydrates. Lectins as histochemical reagents show the presence of a variety of glycoconjugates including mannose, glucose, galactosamine, N-acetyl-galactosamine. Also present were N-acetyl-glucosamine and sialic acid which contribute to the successful establishment and replication or T. cruzi in its insect vectors. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the formation and structure of the PMM is confirmed at 15 days post feeding. Our results confirmed the importance of the feeding processes in the formation of the PMM and showed the nature of the biochemical composition of the vectors' intestine in this important Mexican vector of Chagas disease.

Ecological connectivity of Trypanosoma cruzi reservoirs and Triatoma pallidipennis hosts in an anthropogenic landscape with endemic Chagas disease.

Traditional methods for Chagas disease prevention are targeted at domestic vector reduction, as well as control of transfusion and maternal-fetal transmission. Population connectivity of Trypanosoma cruzi-infected vectors and hosts, among sylvatic, ecotone and domestic habitats could jeopardize targeted efforts to reduce human exposure. This connectivity was evaluated in a Mexican community with reports of high vector infestation, human infection, and Chagas disease, surrounded by agricultural and natural areas. We surveyed bats, rodents, and triatomines in dry and rainy seasons in three adjacent habitats (domestic, ecotone, sylvatic), and measured T. cruzi prevalence, and host feeding sources of triatomines. Of 12 bat and 7 rodent species, no bat tested positive for T. cruzi, but all rodent species tested positive in at least one season or habitat. Highest T. cruzi infection prevalence was found in the rodents, Baiomys musculus and Neotoma mexicana. In general, parasite prevalence was not related to habitat or season, although the sylvatic habitat had higher infection prevalence than by chance, during the dry season. Wild and domestic mammals were identified as bloodmeals of T. pallidipennis, with 9% of individuals having mixed human (4.8% single human) and other mammal species in bloodmeals, especially in the dry season; these vectors tested >50% positive for T. cruzi. Overall, ecological connectivity is broad across this matrix, based on high rodent community similarity, vector and T. cruzi presence. Cost-effective T. cruzi, vector control strategies and Chagas disease transmission prevention will need to consider continuous potential for parasite movement over the entire landscape. This study provides clear evidence that these strategies will need to include reservoir/host species in at least ecotones, in addition to domestic habitats.

Identification of blood meal source and infection with Trypanosoma cruzi of Chagas disease vectors using a multiplex cytochrome b polymerase chain reaction assay.

Long-term control of triatomine bugs in Chagas endemic regions will depend on a full understanding of vector-parasite-host interactions. Herein we describe a cytochrome b multiplex polymerase chain reaction (PCR)-based strategy for blood meal source identification in bug foregut contents. This technique discriminates human from animal blood, and has been tested in five Triatoma species from México. Host identification has been validated for human, four rodent species, two bat species, dog, rabbit, sheep, and opossum. In addition, Trypanosoma cruzi can be identified simultaneously using S34/S67-specific kinetoplast DNA primers. Both host and parasite identification were possible as long as 10 weeks after bug feeding, and in samples stored up to 6 years. The blood meal identification procedure described here represents a powerful tool for large-scale studies identifying the biological, ecological, and environmental variables associated with Chagas disease transmission.