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Trypanosoma rangeli is a non-pathogenic protozoan parasite that infects mammals, including humans, in Chagas disease-endemic areas of South and Central America. The parasite is transmitted to a mammalian host when an infected triatomine injects metacyclic trypomastigotes into the host′s skin during a bloodmeal. Infected mammals behave as parasite reservoirs for several months and despite intensive research, some major aspects of T. rangeli-vertebrate interactions are still poorly understood. In particular, many questions still remain unanswered, e.g. parasite survival and development inside vertebrates, as no parasite multiplication sites have yet been identified. The present study used an insect bite transmission strategy to investigate whether the vector inoculation spot in the skin behave as a parasite-replication site. Histological data from the skin identified extracellular parasites in the dermis and hypodermis of infected mice in the first 24 hours post-infection, as well as the presence of inflammatory infiltrates in a period of up to 7 days. However, qPCR analyses demonstrated that T. rangeli is eliminated from the skin after 7 days of infection despite being still consistently found on circulating blood and secondary lymphoid tissues for up to 30 days post-infection. Interestingly, significant numbers of parasites were found in the spleen and mesenteric lymph nodes of infected mice during different periods of infection and steady basal numbers of flagellates are maintained in the host′s bloodstream, which might behave as a transmission source to insect vectors. The presence of parasites in the spleen was confirmed by fluorescent photomicrography of free and cell-associated T. rangeli forms. Altogether our results suggest that this organ could possibly behave as a T. rangeli maintenance hotspot in vertebrates., Author summary Trypanosoma rangeli development inside vertebrates is a highly controversial field of study, since there is no evidence of parasite multiplication in host tissues. One of the difficulties that turns it even more complex, it’s the challenge to maintain T. rangeli infectivity, since its maintenance in culture decreases parasite’s ability to infect its hosts. Our group developed a methodology in which parasites are kept in frequent passages through triatomines and mice, assuring its infectivity and allowing the development of studies more similar to natural transmission. In this study we used qPCR, flow cytometry and histological techniques to evaluate parasite persistence in different tissues and organs of mice infected by the bite of infected bugs, mimicking a natural infection. We found that after one week of infection, parasites as well as inflammatory infiltrates disappear from the inoculation site at the host´s skin. Evaluation of infected mice for a period of 30 days demonstrated that a stable basal amount of parasites was detected in circulating blood, although an increased amount of T. rangeli DNA was found in mesenteric lymph nodes and spleen. In spleen, through marking the parasite with a monoclonal antibody we confirmed the presence of live parasites which were seen in apparent close association with cells and also inside them as apparently amastigote forms, indicating that the parasite is preserved and possibly replicates in these tissues. Our study provides new insights into the interaction of T. rangeli with its vertebrate hosts.

The acyl-CoA-binding proteins (ACBP) act by regulating the availability of acyl-CoA in the cytoplasm and must have essential functions in lipid metabolism. The genome of the kissing-bug Rhodnius prolixus encodes five proteins of this family, but little is known about them. In this study we investigated the expression and function of RpACBP-5. Feeding induced RpACBP-5 gene expression in the posterior midgut, and an increase of about four times was observed two days after the blood meal. However, the amount of protein, which was only detected in this organ, did not change during digestion. The RpACBP-5 gene was also highly expressed in pre-vitellogenic and vitellogenic oocytes. Recombinant RpACBP-5 was shown to bind to acyl-CoA of different lengths, and it exhibited nanomolar affinity to lauroyl-CoA in an isothermal titration assay, indicating that RpACBP-5 is a functional ACBP. RpACBP-5 knockdown by RNA interference did not affect digestion, egg laying and hatching, survival, or accumulation of triacylglycerol in the fat body and oocytes. Similarly, double knockdown of RpACBP-1 and RpACBP-5 did not alter egg laying and hatching, survival, accumulation of triacylglycerol in the fat body and oocytes, or the neutral lipid composition of the posterior midgut or hemolymph. These results show that RpACBP-5 is a functional ACBP but indicate that the lack of a detectable phenotype in the knockdown insects may be a consequence of functional overlap of the proteins of the ACBP family found in the insect.

The piRNA pathway is a surveillance system that guarantees oogenesis and adult fertility in a range of animal species. The pathway is centered on PIWI clade Argonaute proteins and the associated small non-coding RNAs termed piRNAs. In this study, we set to investigate the evolutionary conservation of the piRNA pathway in the hemimetabolous insect Rhodnius prolixus. Our transcriptome profiling reveals that core components of the pathway are expressed during previtellogenic stages of oogenesis. Rhodnius’ genome harbors four putative piwi orthologs. We show that Rp-piwi2, Rp-piwi3 and Rp-ago3, but not Rp-piwi1 transcripts are produced in the germline tissues and maternally deposited in the mature eggs. Consistent with a role in Rhodnius oogenesis, parental RNAi against the Rp-piwi2, Rp-piwi3 and Rp-ago3 results in severe egg laying and female adult fertility defects. Furthermore, we show that the reduction of the Rp-piwi2 levels by parental RNAi disrupts oogenesis by causing a dramatic loss of trophocytes, egg chamber degeneration and oogenesis arrest. Intriguingly, the putative Rp-Piwi2 protein features a polyglutamine tract at its N-terminal region, which is conserved in PIWI proteins encoded in the genome of other Triatomine species. Together with R. prolixus, these hematophagous insects are primary vectors of the Chagas disease. Thus, our data shed more light on the evolution of the piRNA pathway and provide a framework for the development of new control strategies for Chagas disease insect vectors., Author summary Rhodnius prolixus together with other blood-feeding bugs of the Triatominae family are primary vectors of the protozoan Trypanosoma cruzi, the causative agent of the Chagas disease. It has been estimated that 7–8 million people are affected by this life-threatening illness worldwide, which makes the Chagas disease one of the most neglected tropical diseases. In this study, we describe the transcriptome of previtellogenic stages of Rhodnius oogenesis. Furthermore, by using a combination of molecular biology techniques and functional analyses we show that central components of the piRNA pathway are conserved in this species. The piRNA pathway guarantees genomic stability in the germ cells of organisms as distant as flies and mice. In accordance, we find that the knock-down of the piwi genes, which form the backbone of the pathway, results in partial or complete female adult sterility in Rhodnius. Our data will help improve the annotation of the Rhodnius genome and provide a framework for the development of novel techniques aiming at the eradication of Rhodnius prolixus and other Triatomine species from the infested areas. The achievement of this goal will ultimately prevent the transmission of trypanosomes to humans and reduce or eliminate the diffusion of the Chagas disease.

Background Although the central coast of the Ecuador is considered endemic for Chagas disease, few studies have focused on determining the risk of transmission in this region. In this study we describe the triatomine household infestation in Manabí province (Central Coast region), determine the rate of Trypanosoma cruzi infection and study the risk factors associated with infestation by Rhodnius ecuadoriensis. Methodology/Principal findings An entomological survey found three triatomine species (Rhodnius ecuadoriensis, Panstrongylus rufotuberculatus and P. howardi) infesting domiciles in 47.4% of the 78 communities visited (total infestation rate of 4.5%). Four percent of domiciles were infested, and nymphs were observed in 77% of those domiciles. The three species were found in altitudes below 500 masl and in all ecological zones except cloud forest. Within the domicile, we found the three species mostly in bedrooms. Rhodnius ecuadoriensis and P. rufotuberculatus were abundant in bird nests, including chicken coops and P. howardi associated with rats in piles of bricks, in the peridomicile. Triatomine infestation was characterized by high rates of colonization, especially in peridomicile. Flagelates infection was detected in only 12% of the samples by microscopy and Trypanosoma cruzi infection in 42% of the examined triatomines by PCR (n = 372). The most important risk factors for house infestation by R. ecuadoriensis were ecological zone (w = 0.99) and presence of chickens (w = 0.96). Determinants of secondary importance were reporting no insecticide applications over the last twelve months (w = 0.86) and dirt floor (w = 0.70). On the other hand, wood as wall material was a protective factor (w = 0.85). Conclusion/Significance According the results, approximately 571,000 people would be at high risk for T. cruzi infection in Manabí province. A multidisciplinary approximation and the adhesion to a periodic integrated vector management (IVM) program are essential to guarantee sustainable preventive and control strategies for Chagas disease in this region., Author summary Chagas disease is an important public health problem in most of Latin America, including Ecuador. It is caused by the parasite Trypanosoma cruzi, which in endemic areas is transmitted mainly by the feces of insects called triatomines. Control efforts in the region have reduced the number of new cases of Chagas disease, but there is limited information in certain countries, such as Ecuador. We conducted the first province-wide entomological survey conducted in central coastal Ecuador and report the distribution of triatomines, their habitat and microhabitat preferences, and factors that favor their presence. The results of the study show that 4.5% of households in 78 rural communities harbor triatomine populations. Infestation is higher outside, than inside the houses, but is well established, as shown by the presence of inmature stages and eggs. These results, combined with recent reports of lack of effectiveness of insecticide spraying efforts in this region show the need for innovative approaches for vector control to reduce the high risk of infection to approximately 571,000 people living in rural areas of central coastal Ecuador.

Background Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. Methodology/Principal findings The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. Conclusions/Significance Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment., Author summary Understanding Chagas disease vector (triatomine) population dispersal is key for the design of control measures tailored for the epidemiological situation of a particular region. In Ecuador, Rhodnius ecuadoriensis is a cause of concern for Chagas disease transmission, since it is widely distributed from the central coast to southern Ecuador. Here, a genome-wide sequencing (2b-RAD) approach was performed in 20 specimens from four communities from Manabí (central coast) and Loja (southern) provinces of Ecuador, and the effectiveness of three type IIB restriction enzymes was assessed. The findings of this study show that this genotyping methodology is cost effective in R. ecuadoriensis and likely in other triatomine species. In addition, preliminary population genomic analysis results detected a signal of population structure among geographically distinct communities and genetic variability within communities. As such, 2b-RAD shows significant promise as a relatively low-tech solution for determination of vector population genomics, dynamics, and spread.

Background Chagas disease is endemic to the southern Andean region of Ecuador, an area with one of the highest poverty rates in the country. However, few studies have looked into the epidemiology, vectors and transmission risks in this region. In this study we describe the triatomine household infestation in Loja province, determine the rate of Trypanosoma cruzi infection in triatomines and study the risk factors associated with infestation. Methodology/Principal Findings An entomological survey found four triatomine species (Rhodnius ecuadoriensis, Triatoma carrioni, Panstrongylus chinai, and P. rufotuberculatus) infesting domiciles in 68% of the 92 rural communities examined. Nine percent of domiciles were infested, and nymphs were observed in 80% of the infested domiciles. Triatomines were found in all ecological regions below 2,200 masl. We found R. ecuadoriensis (275 to 1948 masl) and T. carrioni (831 to 2242 masl) mostly in bedrooms within the domicile, and they were abundant in chicken coops near the domicile. Established colonies of P. chinai (175 to 2003 masl) and P. rufotuberculatus (404 to 1613 masl) also were found in the domicile. Triatomine infestation was associated with surrogate poverty indicators, such as poor sanitary infrastructure (lack of latrine/toilet [w = 0.95], sewage to environment [w = 1.0]). Vegetation type was a determinant of infestation [w = 1.0] and vector control program insecticide spraying was a protective factor [w = 1.0]. Of the 754 triatomines analyzed, 11% were infected with Trypanosoma cruzi and 2% were infected with T. rangeli. Conclusions/Significance To date, only limited vector control efforts have been implemented. Together with recent reports of widespread sylvatic triatomine infestation and frequent post-intervention reinfestation, these results show that an estimated 100,000 people living in rural areas of southern Ecuador are at high risk for T. cruzi infection. Therefore, there is a need for a systematic, sustained, and monitored vector control intervention that is coupled with improvement of socio-economic conditions., Author Summary Chagas disease is a major health problem in Latin America. Its infectious agent, Trypanosoma cruzi, is transmitted mainly by the feces of insects called triatomines. Although great progress has been made in reducing transmission in the countries in the southern cone of South America, limited progress has been made in the Andean countries, partly because of lack of knowledge and partly because of limited resources and technical capability of control efforts. We report the first entomological survey conducted in southern Ecuador to learn the distribution of triatomines, their habitat and microhabitat preferences, and factors that favor their presence. The results of the study show that 9% of households in 92 rural communities harbor well-established triatomine populations. These rural areas in the southern Andean region are among the poorest in the country; most of the population lives in sub-standard housing, under conditions that favor the presence of Chagas disease vectors. These results, combined with recent reports of widespread triatomine infestation of wild animal nests in sylvatic areas throughout the region and frequent reports of reinfestation after control intervention, indicate the need for effective, monitored, and sustained vector control to reduce the high risk of infection to which approximately 100,000 people living in rural areas of southern Ecuador are exposed.

Haematophagous insects can ingest large quantities of blood in a single meal and eliminate high volumes of urine in the next few hours. This rise in diuresis is possible because the excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of intensification of aorta contractions combined with an increase of the anterior midgut peristaltic waves. It has been previously described that haemolymph circulation during post-prandial diuresis is stimulated by the synergistic activity of allatotropin (AT) and serotonin in the kissing bug Triatoma infestans; resulting in an increase in aorta contractions. In the same species, AT stimulates anterior midgut and rectum muscle contractions to mix urine and feces and facilitate the voiding of the rectum. Furthermore, levels of AT in midgut and Malpighian tubules increased in the afternoon when insects are getting ready for nocturnal feeding. In the present study we describe the synergistic effect of AT and serotonin increasing the frequency of contractions of the aorta in Rhodnius prolixus. The basal frequency of contractions of the aorta in the afternoon is higher that the observed during the morning, suggesting the existence of a daily rhythmic activity. The AT receptor is expressed in the rectum, midgut and dorsal vessel, three critical organs involved in post-randial diuresis. All together these findings provide evidence that AT plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus., Facultad de Ciencias Naturales y Museo

Peptidergic signaling regulates cardiac contractility; thus, identifying molecular switches, ligand-receptor contacts, and antagonists aids in exploring the underlying mechanisms to influence health. Myosuppressin (MS), a decapeptide, diminishes cardiac contractility and gut motility. Myosuppressin binds to G protein-coupled receptor (GPCR) proteins. Two Drosophila melanogaster myosuppressin receptors (DrmMS-Rs) exist; however, no mechanism underlying MS-R activation is reported. We predicted DrmMS-Rs contained molecular switches that resembled those of Rhodopsin. Additionally, we believed DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 interactions would reflect our structure-activity relationship (SAR) data. We hypothesized agonist- and antagonist-receptor contacts would differ from one another depending on activity. Lastly, we expected our study to apply to other species; we tested this hypothesis in Rhodnius prolixus, the Chagas disease vector. Searching DrmMS-Rs for molecular switches led to the discovery of a unique ionic lock and a novel 3-6 lock, as well as transmission and tyrosine toggle switches. The DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contacts suggested tissue-specific signaling existed, which was in line with our SAR data. We identified R. prolixus (Rhp)MS-R and discovered it, too, contained the unique myosuppressin ionic lock and novel 3-6 lock found in DrmMS-Rs as well as transmission and tyrosine toggle switches. Further, these motifs were present in red flour beetle, common water flea, honey bee, domestic silkworm, and termite MS-Rs. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM. Based on ligand-receptor contacts, we designed RhpMS analogs believed to be an active core and antagonist; testing on heart confirmed these predictions. The active core docking mimicked RhpMS, however, the antagonist did not. Together, these data were consistent with the unique ionic lock, novel 3-6 lock, transmission switch, and tyrosine toggle switch being involved in mechanisms underlying TM movement and MS-R activation, and the ability of MS agonists and antagonists to influence physiology., Facultad de Ciencias Exactas

Background Chagas disease is a serious public health problem in Latin America where about ten million individuals show Trypanosoma cruzi infection. Despite significant success in controlling domiciliated triatomines, sylvatic populations frequently infest houses after insecticide treatment which hampers long term control prospects in vast geographical areas where vectorial transmission is endemic. As a key issue, the spatio-temporal dynamics of sylvatic populations is likely influenced by landscape yet evidence showing this effect is rare. The aim of this work is to examine the role of land cover changes in sylvatic triatomine ecology, based on an exhaustive field survey of pathogens, vectors, hosts, and microhabitat characteristics' dynamics. Methodology and Principal Findings The study was performed in agricultural landscapes of coastal Ecuador as a study model. Over one year, a spatially-randomized sampling design (490 collection points) allowed quantifying triatomine densities in natural, cultivated and domestic habitats. We also assessed infection of the bugs with trypanosomes, documented their microhabitats and potential hosts, and recorded changes in landscape characteristics. In total we collected 886 individuals, mainly represented by nymphal stages of one triatomine species Rhodnius ecuadoriensis. As main results, we found that 1) sylvatic triatomines had very high T. cruzi infection rates (71%) and 2) densities of T. cruzi-infected sylvatic triatomines varied predictably over time due to changes in land cover and occurrence of associated rodent hosts. Conclusion We propose a framework for identifying the factors affecting the yearly distribution of sylvatic T. cruzi vectors. Beyond providing key basic information for the control of human habitat colonization by sylvatic vector populations, our framework highlights the importance of both environmental and sociological factors in shaping the spatio-temporal population dynamics of triatomines. A better understanding of the dynamics of such socio-ecological systems is a crucial, yet poorly considered, issue for the long-term control of Chagas disease., Author Summary Globally, more than 10 million people are infected with Trypanosoma cruzi. The emergence and perpetuation of Chagas disease in some endemic countries, such as Ecuador, depends largely on sylvatic populations of T. cruzi-infected vectors that frequently infest houses after insecticide treatment thereby hampering long-term control prospects in vast geographical areas. Our study describes, for the first time in an agricultural landscape, how the temporal dynamics of sylvatic vector, host, and pathogen populations interact spatially in a farming community of coastal Ecuador. In particular, we found that land cover changes due to both farming activities and vegetation phenology affect rodent host distribution and consequently the relative abundance of vectors involved in the transmission cycle of T. cruzi.

The Rhodnius Pacific group is composed of three species: Rhodnius pallescens, R. colombiensis and R. ecuadoriensis, which are considered important vectors of trypanosomes (Trypanosoma cruzi and T. rangeli) infecting humans. This group is considered as a recent trans-Andean lineage derived from the widespread distributed sister taxa R. pictipes during the later uplift of northern Andes mountain range. The widest spread species R. pallescens may be a complex of two divergent lineages with different chromosomal attributes and a particular biogeographical distribution across Central America and Colombia with several southern populations in Colombia occupying the same sylvatic habitat as its sister species R. colombiensis. Although the taxonomy of Rhodnius Pacific group has been well studied, the unresolved phylogenetic and systematic issues are the target of this paper. Here we explore the molecular phylogeography of this species group analyzing two mitochondrial (ND4 and cyt b) and one nuclear (D2 region of ribosomal 28S gene) gene sequences. The molecular analyses suggest an early divergence of the species R. ecuadoriensis and R. colombiensis, followed by a recent expansion of R. pallescens lineages. The phylogenetic relationship between sympatric R. pallescens Colombian lineage and R. colombiensis was further explored using wing morphometry, DNA genome size measurements, and by analyzing chromosomal behavior of hybrids progeny obtained from experimental crosses. Our results suggest that the diversification of the two R. pallescens lineages was mainly influenced by biogeographical events such as (i) the emergence of the Panama Isthmus, while the origin and divergence of R. colombiensis was associated with (ii) the development of particular genetic and chromosomal features that act as isolation mechanisms from its sister species R. pallescens (Colombian lineage). These findings provide new insights into the evolution of the Rhodnius Pacific group and the underlying biological processes that occurred during its divergence.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet., Journal Article, Research Support, N.I.H. Intramural, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Background TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. Methodology and Principal Findings Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. Conclusion and Significance Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus., Author Summary Chagas disease, the major tropical human disease in much of Latin America, affects approximately 11 million people. There are 300,000 new cases of Chagas disease and approximately 21,000 deaths, annually. Triatomine vectors, including Rhodnius prolixus, are able to transmit the protozoan Trypanosoma cruzi, the etiological agent of disease. To develop within insects, the flagellates undergo morphological changes, modulating surface molecules to enable interactions with insect tissues such as the perimicrovilar membranes in the midgut which is an essential step for their development and successful transmission to a vertebrate host. The surface of T. cruzi is covered in glycosyl phosphatidylinositol (GPI)-anchored mucin molecules that determine parasite protection and establishment of a persistent infection in vertebrates. A particular kind of mucin, termed TcSMUG L, is only present at surface of the insect-dwelling stages of protozoan and, according to our results, it is involved in the interaction between T. cruzi and its invertebrate host, determining both the ex vivo adhesion to the insect midgut cells and the in vivo development in the vector. Collectively, our work adds new insight into the relevance of mucin-type glycoconjugates in the infection of insect vectors and points to them as promising targets to develop transmission-blocking strategies for this disease.

The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pKas can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different “microscopic”, conformation-dependent pKa values. In those cases, interpretation of experimental measurements of the pKa is complicated by the coupling between pH, protonation state and protein conformation. We explored these issues using Nitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation where NO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open state from which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pKas: 8.5 in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopic values to the experimentally observed “apparent” pKa, obtaining a value of 6.5, in excellent agreement with experimental data. This value must be interpreted as the pH at which the closed to open population transition takes place. More generally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentally observed ensemble dependent apparent pKas and that the insight gained in the relatively simple case of NP4 can be useful in several more complex cases involving a pH dependent transition, of great biochemical interest., Author Summary The interaction of an amino acid with its protein environment can result in an acid-base behavior that is very different from what would be observed in solution. This environment can be greatly altered when the protein changes conformation. As a result, the amino acid will have two different “microscopic” pKa values. Nitrophorin 4 is a good case study to explore this behavior, because it undergoes a pH-dependent conformational change that is well characterized experimentally. Using computer simulation tools, we found that the key titratable Aspartic acid 30, has two very different microscopic pKas: 4.3 and 8.5, which are significantly different to the observed transition pKa in solution. However, using a simple model, we were able to understand how this causes the conformational change to take place at pH∼6.5, as measured experimentally. The insight gained in this relatively simple case can be useful in other more complex cases where the apparent pKa is also a result of the interplay of different conformations where some amino acids experience very different environments.

BackgroundRhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.Methodology/principal findingsHere we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities.Conclusions/significanceTaken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

Background: The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus. Methodology/Principal findings: Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H(+)-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5 +/- 0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs. Conclusions: We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells., Article, PLOS ONE. 6(11):e27276 (2011)

Background Anthropogenic land use may influence transmission of multi-host vector-borne pathogens by changing diversity, relative abundance, and community composition of reservoir hosts. These reservoir hosts may have varying competence for vector-borne pathogens depending on species-specific characteristics, such as life history strategy. The objective of this study is to evaluate how anthropogenic land use change influences blood meal species composition and the effects of changing blood meal species composition on the parasite infection rate of the Chagas disease vector Rhodnius pallescens in Panama. Methodology/Principal Findings R. pallescens vectors (N = 643) were collected in different habitat types across a gradient of anthropogenic disturbance. Blood meal species in DNA extracted from these vectors was identified in 243 (40.3%) vectors by amplification and sequencing of a vertebrate-specific fragment of the 12SrRNA gene, and T. cruzi vector infection was determined by pcr. Vector infection rate was significantly greater in deforested habitats as compared to contiguous forests. Forty-two different species of blood meal were identified in R. pallescens, and species composition of blood meals varied across habitat types. Mammals (88.3%) dominated R. pallescens blood meals. Xenarthrans (sloths and tamanduas) were the most frequently identified species in blood meals across all habitat types. A regression tree analysis indicated that blood meal species diversity, host life history strategy (measured as rmax, the maximum intrinsic rate of population increase), and habitat type (forest fragments and peridomiciliary sites) were important determinants of vector infection with T. cruzi. The mean intrinsic rate of increase and the skewness and variability of rmax were positively associated with higher vector infection rate at a site. Conclusions/Significance In this study, anthropogenic landscape disturbance increased vector infection with T. cruzi, potentially by changing host community structure to favor hosts that are short-lived with high reproductive rates. Study results apply to potential environmental management strategies for Chagas disease., Author Summary Understanding how host species influence vector-borne pathogen transmission in anthropogenically disturbed landscapes is important to predicting and preventing disease transmission. This study evaluates how host diversity, anthropogenic land use change, and host life history influence vector- borne multihost pathogen transmission in Panama, where the triatomine bug Rhodnius pallescens is the principal vector of Trypanosoma cruzi, agent of Chagas disease. We hypothesize that blood meal species composition and vector infection differ as a function of habitat disturbance, and that the host species intrinsic rate of increase is positively associated with T. cruzi vector infection. We collected R. pallescens across a gradient of anthropogenic disturbance. Blood meal species composition and T. cruzi vector infection were determined by molecular methods. Vector infection rates were higher in deforested habitats and forest fragments as compared to contiguous forests. Vectors fed primarily on mammals, likely accounting for a relatively high vector infection prevalence, and that host blood meal species composition varied across habitat types. Regression tree analysis demonstrates that higher T. cruzi vector infection indices we associated with sites that had blood meal species with higher, more variable, and more skewed rmax (intrinsic rates of increase) values, lower blood meal species diversity, and disturbed habitats, namely fragmented forests and peridomiciliary sites.