Spatial epidemiology and adaptive targeted sampling to manage the Chagas disease vector Triatoma dimidiata.

Widespread application of insecticide remains the primary form of control for Chagas disease in Central America, despite only temporarily reducing domestic levels of the endemic vector Triatoma dimidiata and having little long-term impact. Recently, an approach emphasizing community feedback and housing improvements has been shown to yield lasting results. However, the additional resources and personnel required by such an intervention likely hinders its widespread adoption. One solution to this problem would be to target only a subset of houses in a community while still eliminating enough infestations to interrupt disease transfer. Here we develop a sequential sampling framework that adapts to information specific to a community as more houses are visited, thereby allowing us to efficiently find homes with domiciliary vectors while minimizing sampling bias. The method fits Bayesian geostatistical models to make spatially informed predictions, while gradually transitioning from prioritizing houses based on prediction uncertainty to targeting houses with a high risk of infestation. A key feature of the method is the use of a single exploration parameter, α, to control the rate of transition between these two design targets. In a simulation study using empirical data from five villages in southeastern Guatemala, we test our method using a range of values for α, and find it can consistently select fewer homes than random sampling, while still bringing the village infestation rate below a given threshold. We further find that when additional socioeconomic information is available, much larger savings are possible, but that meeting the target infestation rate is less consistent, particularly among the less exploratory strategies. Our results suggest new options for implementing long-term T. dimidiata control.

Assessing risk of vector transmission of Chagas disease through blood source analysis using LC-MS/MS for hemoglobin sequence identification.

Chagas disease is mainly transmitted by triatomine insect vectors that feed on vertebrate blood. The disease has complex domiciliary infestation patterns and parasite transmission dynamics, influenced by biological, ecological, and socioeconomic factors. In this context, feeding patterns have been used to understand vector movement and transmission risk. Recently, a new technique using Liquid chromatography tandem mass spectrometry (LC-MS/MS) targeting hemoglobin peptides has showed excellent results for understanding triatomines' feeding patterns. The aim of this study was to further develop the automated computational analysis pipeline for peptide sequence taxonomic identification, enhancing the ability to analyze large datasets data. We then used the enhanced pipeline to evaluate the feeding patterns of Triatoma dimidiata, along with domiciliary infestation risk variables, such as unkempt piles of firewood or construction material, cracks in bajareque and adobe walls and intradomiciliary animals. Our new python scripts were able to detect blood meal sources in 100% of the bugs analyzed and identified nine different species of blood meal sources. Human, chicken, and dog were the main blood sources found in 78.7%, 50.4% and 44.8% of the bugs, respectively. In addition, 14% of the bugs feeding on chicken and 15% of those feeding on dogs were captured in houses with no evidence of those animals being present. This suggests a high mobility among ecotopes and houses. Two of the three main blood sources, dog and chicken, were significantly (p < 0.05) affected by domiciliary infestation risk variables, including cracks in walls, construction material and birds sleeping in the intradomicile. This suggests that these variables are important for maintaining reproducing Triatoma dimidiata populations and that it is critical to mitigate these variables in all the houses of a village for effective control of these mobile vectors.

Insights from a comprehensive study of Trypanosoma cruzi: A new mitochondrial clade restricted to North and Central America and genetic structure of TcI in the region.

More than 100 years since the first description of Chagas Disease and with over 29,000 new cases annually due to vector transmission (in 2010), American Trypanosomiasis remains a Neglected Tropical Disease (NTD). This study presents the most comprehensive Trypanosoma cruzi sampling in terms of geographic locations and triatomine species analyzed to date and includes both nuclear and mitochondrial genomes. This addresses the gap of information from North and Central America. We incorporate new and previously published DNA sequence data from two mitochondrial genes, Cytochrome oxidase II (COII) and NADH dehydrogenase subunit 1 (ND1). These T. cruzi samples were collected over a broad geographic range including 111 parasite DNA samples extracted from triatomines newly collected across North and Central America, all of which were infected with T. cruzi in their natural environment. In addition, we present parasite reduced representation (Restriction site Associated DNA markers, RAD-tag) genomic nuclear data combined with the mitochondrial gene sequences for a subset of the triatomines (27 specimens) collected from Guatemala and El Salvador. Our mitochondrial phylogenetic reconstruction revealed two of the major mitochondrial lineages circulating across North and Central America, as well as the first ever mitochondrial data for TcBat from a triatomine collected in Central America. Our data also show that within mtTcIII, North and Central America represent an independent, distinct clade from South America, named here as mtTcIIINA-CA, geographically restricted to North and Central America. Lastly, the most frequent lineage detected across North and Central America, mtTcI, was also an independent, distinct clade from South America, noted as mtTcINA-CA. Furthermore, nuclear genome data based on Single Nucleotide Polymorphism (SNP) showed genetic structure of lineage TcI from specimens collected in Guatemala and El Salvador supporting the hypothesis that genetic diversity at a local scale has a geographical component. Our multiscale analysis contributes to the understanding of the independent and distinct evolution of T. cruzi lineages in North and Central America regions.

Catch me if you can: Under-detection of Trypanosoma cruzi (Kinetoplastea: Trypanosomatida) infections in Triatoma dimidiata s.l. (Hemiptera: Reduviidae) from Central America.

Assays for parasite detection in insect vectors provide important information for disease control. American Trypanosomiasis (Chagas disease) is the most devastating vector-borne illness and the fourth most common in Central America behind HIV/AIDS and acute respiratory and diarrheal infections (Peterson et al., 2019). Under-detection of parasites is a general problem which may be influenced by parasite genetic variation; however, little is known about the genetic variation of the Chagas parasite, especially in this region. In this study we compared six assays for detecting the Chagas parasite, Trypanosoma cruzi: genomic reduced representation sequencing (here referred to as genotype-by-sequencing or GBS), two with conventional PCR (i.e., agarose gel detection), two with qPCR, and microscopy. Our results show that, compared to GBS genomic analysis, microscopy and PCR under-detected T. cruzi in vectors from Central America. Of 94 samples, 44% (50/94) were positive based on genomic analysis. The lowest detection, 9% (3/32) was in a subset assayed with microscopy. Four PCR assays, two with conventional PCR and two with qPCR showed intermediate levels of detection. Both qPCR tests and one conventional PCR test targeted the 195 bp repeat of satellite DNA while the fourth test targeted the 18S gene. Statistical analyses of the genomic and PCR results indicate that the PCR assays significantly under detect infections of Central American T. cruzi genotypes.

From e-voucher to genomic data: Preserving archive specimens as demonstrated with medically important mosquitoes (Diptera: Culicidae) and kissing bugs (Hemiptera: Reduviidae).

Scientific collections such as the U.S. National Museum (USNM) are critical to filling knowledge gaps in molecular systematics studies. The global taxonomic impediment has resulted in a reduction of expert taxonomists generating new collections of rare or understudied taxa and these large historic collections may be the only reliable source of material for some taxa. Integrated systematics studies using both morphological examinations and DNA sequencing are often required for resolving many taxonomic issues but as DNA methods often require partial or complete destruction of a sample, there are many factors to consider before implementing destructive sampling of specimens within scientific collections. We present a methodology for the use of archive specimens that includes two crucial phases: 1) thoroughly documenting specimens destined for destructive sampling-a process called electronic vouchering, and 2) the pipeline used for whole genome sequencing of archived specimens, from extraction of genomic DNA to assembly of putative genomes with basic annotation. The process is presented for eleven specimens from two different insect subfamilies of medical importance to humans: Anophelinae (Diptera: Culicidae)-mosquitoes and Triatominae (Hemiptera: Reduviidae)-kissing bugs. Assembly of whole mitochondrial genome sequences of all 11 specimens along with the results of an ortholog search and BLAST against the NCBI nucleotide database are also presented.

Infestation dynamics of Triatoma dimidiata in highly deforested tropical dry forest regions of Guatemala.

BACKGROUND: Deforestation, driven by anthropogenic change in land use, influences the behaviour and abundance of vector-borne diseases. For various species of Chagas disease vectors, there is evidence that change in land use affects population density and abundance. Triatoma dimidiata is the most important Chagas vector in Guatemala, and at least one million people live in T. dimidiata endemic areas; however, infestation dynamics vary among regions, from high infestation with all life stages to low seasonal infestation by sylvatic adults. OBJECTIVES: The aim of this study was to evaluate how land-use, combined with domiciliary risk factors, influences the infestation dynamics of T. dimidiata for four villages in a dry forest region with a strong deforestation history. METHODS: Land use, measured with drone and satellite images, was classified into four categories (houses, monocultures and pastures, woodland and shrubland, and bare soil). Domiciliary risk factors and infestation were assessed through entomological surveys. Statistical analyses compared infestation indices and the ability of land use and domiciliary risk factors to explain infestation. FINDINGS: Two villages had significantly higher infestation (26 and 30% vs. 5 and 6%), yet all villages had high colonisation (71-100% of infested houses had immature insects), with no significant difference among them. Because of the high level of deforestation across the study area, land use was not related to infestation; however, domiciliary risk factors were. A model based on four weighted domiciliary risk factors (adobe or bajareque walls, intradomicile animals, intradomicile clutter, and dirt floors) explains the infestation risk. MAIN CONCLUSIONS: Because almost all infested houses have reproducing populations in this deforested dry forest region and statistical analysis identified the domiciliary risk factors for infestation, intermediate and long-term control of Chagas disease vectors in this region requires management of these risk factors.

Novel Evolutionary Algorithm Identifies Interactions Driving Infestation of Triatoma dimidiata, a Chagas Disease Vector.

Chagas disease is a lethal, neglected tropical disease. Unfortunately, aggressive insecticide-spraying campaigns have not been able to eliminate domestic infestation of Triatoma dimidiata, the native vector in Guatemala. To target interventions toward houses most at risk of infestation, comprehensive socioeconomic and entomologic surveys were conducted in two towns in Jutiapa, Guatemala. Given the exhaustively large search space associated with combinations of risk factors, traditional statistics are limited in their ability to discover risk factor interactions. Two recently developed statistical evolutionary algorithms, specifically designed to accommodate risk factor interactions and heterogeneity, were applied to this large combinatorial search space and used in tandem to identify sets of risk factor combinations associated with infestation. The optimal model includes 10 risk factors in what is known as a third-order disjunctive normal form (i.e., infested households have chicken coops AND deteriorated bedroom walls OR an accumulation of objects AND dirt floors AND total number of occupants ≥ 5 AND years of electricity ≥ 5 OR poor hygienic condition ratings AND adobe walls AND deteriorated walls AND dogs). Houses with dirt floors and deteriorated walls have been reported previously as risk factors and align well with factors currently targeted by Ecohealth interventions to minimize infestation. However, the tandem evolutionary algorithms also identified two new socioeconomic risk factors (i.e., households having many occupants and years of electricity ≥ 5). Identifying key risk factors may help with the development of new Ecohealth interventions and/or reduce the survey time needed to identify houses most at risk.

Infestation dynamics of Triatoma dimidiata in highly deforested tropical dry forest regions of Guatemala

BACKGROUND Deforestation, driven by anthropogenic change in land use, influences the behaviour and abundance of vector-borne diseases. For various species of Chagas disease vectors, there is evidence that change in land use affects population density and abundance. Triatoma dimidiata is the most important Chagas vector in Guatemala, and at least one million people live in T. dimidiata endemic areas; however, infestation dynamics vary among regions, from high infestation with all life stages to low seasonal infestation by sylvatic adults. OBJECTIVES The aim of this study was to evaluate how land-use, combined with domiciliary risk factors, influences the infestation dynamics of T. dimidiata for four villages in a dry forest region with a strong deforestation history. METHODS Land use, measured with drone and satellite images, was classified into four categories (houses, monocultures and pastures, woodland and shrubland, and bare soil). Domiciliary risk factors and infestation were assessed through entomological surveys. Statistical analyses compared infestation indices and the ability of land use and domiciliary risk factors to explain infestation. FINDINGS Two villages had significantly higher infestation (26 and 30% vs. 5 and 6%), yet all villages had high colonisation (71-100% of infested houses had immature insects), with no significant difference among them. Because of the high level of deforestation across the study area, land use was not related to infestation; however, domiciliary risk factors were. A model based on four weighted domiciliary risk factors (adobe or bajareque walls, intradomicile animals, intradomicile clutter, and dirt floors) explains the infestation risk. MAIN CONCLUSIONS Because almost all infested houses have reproducing populations in this deforested dry forest region and statistical analysis identified the domiciliary risk factors for infestation, intermediate and long-term control of Chagas disease vectors in this region requires management of these risk factors.

Protein mass spectrometry detects multiple bloodmeals for enhanced Chagas disease vector ecology.

Chagas disease, a neglected tropical disease endemic in Latin America, is caused by the protozoan parasite Trypanosoma cruzi and is responsible for significant health impacts, especially in rural communities. The parasite is transmitted by insect vectors in the Triatominae subfamily and due to lack of vaccines and limited treatment options, vector control is the main way of controlling the disease. Knowing what vectors are feeding on directly enhances our understanding of the ecology and biology of the different vector species and can potentially aid in engaging communities in active disease control, a concept known as Ecohealth management. We evaluated bloodmeals in rural community, house-caught insect vectors previously evaluated for bloodmeals via DNA analysis as part of a larger collaborative project from three countries in Central America, including Guatemala. In addition to identifying bloodmeals in 100% of all samples using liquid chromatography tandem mass spectrometry (LC-MS/MS) (n = 50), strikingly for 53% of these samples there was no evidence of a recent bloodmeal by DNA-PCR. As individual vectors often feed on multiple sources, we developed an enhanced detection pipeline, and showed the ability to quantify a bloodmeal using stable-isotope-containing synthetic references peptides, a first step in further exploration of species-specific bloodmeal composition. Furthermore, we show that a lower resolution mass spectrometer is sufficient to correctly identify taxa from bloodmeals, an important and strong attribute of our LC-MS/MS-based method, opening the door to using proteomics in countries where Chagas disease is endemic.

Residual survival and local dispersal drive reinfestation by Triatoma dimidiata following insecticide application in Guatemala.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and transmitted by triatomine insect vectors. In Guatemala, insecticide spraying is an integral part of management of the main vector, Triatoma dimidiata. Spraying typically has low efficacy, which may be due to incomplete elimination from infested houses, within-village dispersal, or influx from other villages or sylvan environments. To evaluate how these mechanisms contribute to reinfestation, we conducted a time-course analysis of T. dimidiata infestation, abundance and household genetic structure in two nearby villages in Jutiapa, Guatemala; houses in the first village were surveyed, treated with insecticide if infested and then re-surveyed at eight and 22 months following spraying, while the second village served as an untreated control to quantify changes associated with seasonal dispersal. Insects were genotyped at 2-3000 SNP loci for kinship and population genetic analyses. Insecticide application reduced overall infestation and abundance, while the untreated village was stable over time. Nevertheless, within two years 35.5% of treated houses were reinfested and genetic diversity had largely recovered. Insects collected from reinfested houses post-spraying were most closely related to pre-spray collections from the same house, suggesting that infestations had not been fully eliminated. Immigration by unrelated insects was also detected within a year of spraying; when it occurred, dispersal was primarily local from neighboring houses. Similar dispersal patterns were observed following the annual dispersal season in the untreated village, with high-infestation houses serving as sources for neighboring homes. Our findings suggest that the efficacy of pyrethroid application is rapidly diminished by both within-house breeding by survivors and annual cycles of among-house movement. Given these patterns, we conclude that house structural improvements, an integral part of the Ecohealth approach that makes houses refractory to vector colonization and persistence, are critical for long-term reduction of T. dimidiata infestation.

Description of Triatomahuehuetenanguensis sp. n., a potential Chagas disease vector (Hemiptera, Reduviidae, Triatominae).

A new species of the genus Triatoma Laporte, 1832 (Hemiptera, Reduviidae) is described based on specimens collected in the department of Huehuetenango, Guatemala. Triatomahuehuetenanguensis sp. n. is closely related to T.dimidiata (Latreille, 1811), with the following main morphological differences: lighter color; smaller overall size, including head length; and width and length of the pronotum. Natural Trypanosomacruzi (Chagas, 1909) infection, coupled with its presence in domestic habitats, makes this species a potentially important vector of Trypanosomacruzi in Guatemala.

Implementation science: Epidemiology and feeding profiles of the Chagas vector Triatoma dimidiata prior to Ecohealth intervention for three locations in Central America.

The Ecohealth strategy is a multidisciplinary data-driven approach used to improve the quality of people's lives in Chagas disease endemic areas, such as regions of Central America. Chagas is a vector-borne disease caused by the parasite Trypanosoma cruzi. In Central America, the main vector is Triatoma dimidiata. Because successful implementation of the Ecohealth approach reduced home infestation in Jutiapa department, Guatemala, it was scaled-up to three localities, one in each of three Central American countries (Texistepeque, El Salvador; San Marcos de la Sierra, Honduras and Olopa, Guatemala). As a basis for the house improvement phase of the Ecohealth program, we determined if the localities differ in the role of sylvatic, synanthropic and domestic animals in the Chagas transmission cycle by measuring entomological indices, blood meal sources and parasite infection from vectors collected in and around houses. The Polymerase Chain Reaction (PCR) with taxa specific primers to detect both, blood sources and parasite infection, was used to assess 71 T. dimidiata from Texistepeque, 84 from San Marcos de la Sierra and 568 from Olopa. Our results show that infestation (12.98%) and colonization (8.95%) indices were highest in Olopa; whereas T. cruzi prevalence was higher in Texistepeque and San Marcos de la Sierra (>40%) than Olopa (8%). The blood meal source profiles showed that in Olopa, opossum might be important in linking the sylvatic and domestic Chagas transmission cycle, whereas in San Marcos de la Sierra dogs play a major role in maintaining domestic transmission. For Texistepeque, bird was the major blood meal source followed by human. When examining the different life stages, we found that in Olopa, the proportion bugs infected with T. cruzi is higher in adults than nymphs. These findings highlight the importance of location-based recommendations for decreasing human-vector contact in the control of Chagas disease.

Uncovering vector, parasite, blood meal and microbiome patterns from mixed-DNA specimens of the Chagas disease vector Triatoma dimidiata.

Chagas disease, considered a neglected disease by the World Health Organization, is caused by the protozoan parasite Trypanosoma cruzi, and transmitted by >140 triatomine species across the Americas. In Central America, the main vector is Triatoma dimidiata, an opportunistic blood meal feeder inhabiting both domestic and sylvatic ecotopes. Given the diversity of interacting biological agents involved in the epidemiology of Chagas disease, having simultaneous information on the dynamics of the parasite, vector, the gut microbiome of the vector, and the blood meal source would facilitate identifying key biotic factors associated with the risk of T. cruzi transmission. In this study, we developed a RADseq-based analysis pipeline to study mixed-species DNA extracted from T. dimidiata abdomens. To evaluate the efficacy of the method across spatial scales, we used a nested spatial sampling design that spanned from individual villages within Guatemala to major biogeographic regions of Central America. Information from each biotic source was distinguished with bioinformatics tools and used to evaluate the prevalence of T. cruzi infection and predominant Discrete Typing Units (DTUs) in the region, the population genetic structure of T. dimidiata, gut microbial diversity, and the blood meal history. An average of 3.25 million reads per specimen were obtained, with approximately 1% assigned to the parasite, 20% to the vector, 11% to bacteria, and 4% to putative blood meals. Using a total of 6,405 T. cruzi SNPs, we detected nine infected vectors harboring two distinct DTUs: TcI and a second unidentified strain, possibly TcIV. Vector specimens were sufficiently variable for population genomic analyses, with a total of 25,710 T. dimidiata SNPs across all samples that were sufficient to detect geographic genetic structure at both local and regional scales. We observed a diverse microbiotic community, with significantly higher bacterial species richness in infected T. dimidiata abdomens than those that were not infected. Unifrac analysis suggests a common assemblage of bacteria associated with infection, which co-occurs with the typical gut microbial community derived from the local environment. We identified vertebrate blood meals from five T. dimidiata abdomens, including chicken, dog, duck and human; however, additional detection methods would be necessary to confidently identify blood meal sources from most specimens. Overall, our study shows this method is effective for simultaneously generating genetic data on vectors and their associated parasites, along with ecological information on feeding patterns and microbial interactions that may be followed up with complementary approaches such as PCR-based parasite detection, 18S eukaryotic and 16S bacterial barcoding.

Protein mass spectrometry extends temporal blood meal detection over polymerase chain reaction in mouse-fed Chagas disease vectors.

BACKGROUND: Chagas disease is highly prevalent in Latin America, and vector control is the most effective control strategy to date. We have previously shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) is a valuable tool for identifying triatomine vector blood meals. OBJECTIVES: The purpose of this study was to determine blood meal detection ability as a function of method [polymerase chain reaction (PCR) vs. LC-MS/MS], time since feeding, and the effect of molting in mouse-fed triatomine insect vectors targeting hemoglobin and albumin proteins with LC-MS/MS and short interspersed nuclear elements (SINE)-based PCR. METHODS: We experimentally fed Triatoma protracta on mice and used LC-MS/MS to detect hemoglobin and albumin peptides over time post-feeding and post-molting (≤ 12 weeks). We compared LC-MS/MS results with those of a standard PCR method based on SINEs. FINDINGS: Hemoglobin-based LC-MS/MS detected blood meals most robustly at all time points post-feeding. Post-molting, no blood meals were detected with PCR, whereas LC-MS/MS detected mouse hemoglobin and albumin up to 12 weeks. MAIN CONCLUSIONS: In our study, the hemoglobin signature in the insect abdomen lasted longer than that of albumin and DNA. LC-MS/MS using hemoglobin shows promise for identifying triatomine blood meals over long temporal scales and even post-molting. Clarifying the frequency of blood-feeding on different hosts can foster our understanding of vector behavior and may help devise sounder disease-control strategies, including Ecohealth (community based ecosystem management) approaches.

Description of Triatoma mopan sp. n. from a cave in Belize (Hemiptera, Reduviidae, Triatominae).

In this paper, Triatoma mopansp. n. is described based on five males and six females collected in the Rio Frio cave, Cayo District, Belize. This species is similar to Triatoma dimidiata (Latreille), but can be distinguished by characters found on the pronotum, legs, and abdomen. Geometric morphometry and phylogenetic comparisons are also provided. Presently, the species is known only from the type locality and is a potential Chagas vector.

The role of natural selection in shaping genetic variation in a promising Chagas disease drug target: Trypanosoma cruzi trans-sialidase.

Rational drug design creates innovative therapeutics based on knowledge of the biological target to provide more effective and responsible therapeutics. Chagas disease, endemic throughout Latin America, is caused by Trypanosoma cruzi, a protozoan parasite. Current therapeutics are problematic with widespread calls for new approaches. Researchers are using rational drug design for Chagas disease and one target receiving considerable attention is the T. cruzi trans-sialidase protein (TcTS). In T. cruzi, trans-sialidase catalyzes the transfer of sialic acid from a mammalian host to coat the parasite surface membrane and avoid immuno-detection. However, the role of TcTS in pathology variance among and within genetic variants of the parasite is not well understood despite numerous studies. Previous studies reported the crystalline structure of TcTS and the TS protein structure in other trypanosomes where the enzyme is often inactive. However, no study has examined the role of natural selection in genetic variation in TcTS. To understand the role of natural selection in TcTS DNA sequence and protein variation, we examined a 471 bp portion of the TcTS gene from 48 T. cruzi samples isolated from insect vectors. Because there may be multiple parasite genotypes infecting one insect and there are multiple copies of TcTS per parasite genome, all 48 sequences had multiple polymorphic bases. To resolve these polymorphisms, we examined cloned sequences from two insect vectors. The data are analyzed to understand the role of natural selection in shaping genetic variation in TcTS and interpreted in light of the possible role of TcTS as a drug target. The analysis highlights negative or purifying selection on three amino acids previously shown to be important in TcTS transfer activity. One amino acid in particular, Tyr342, is a strong candidate for a drug target because it is under negative selection and amino acid substitutions inactivate TcTS transfer activity. AUTHOR SUMMARY: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and transmitted to humans and other mammals primarily by Triatomine insects. Being endemic in many South and Central American countries and affecting millions of people the need for new more effective and safe therapies is evident. Here, we examine genetic variation and natural selection on DNA (471 bp) and amino acid (157 aa) sequence data of the T. cruzi trans-sialdiase (TcTS) protein, often suggested as a candidate for rational drug design. In our surveyed region of the protein there were five amino acid residues that have been shown to be integral to the function of TcTS. We found that three were under strong negative selection making them ideal candidates for drug design; however, one was under balancing selection and should be avoided as a drug target. Our study provides new information into identifying potential targets for a new Chagas drug.

Vectors of diversity: Genome wide diversity across the geographic range of the Chagas disease vector Triatoma dimidiata sensu lato (Hemiptera: Reduviidae).

To date, the phylogeny of Triatoma dimidiata sensu lato (s. l.) (Hemiptera: Reduviidae: Triatominae), the epidemiologically most important Chagas disease vector in Central America and a secondary vector in Mexico and northern South America, has only been investigated by one multi-copy nuclear gene (Internal Transcribed Spacer - 2) and a few mitochondrial genes. We examined 450 specimens sampled across most of its native range from Mexico to Ecuador using reduced representation next-generation sequencing encompassing over 16,000 single nucleotide polymorphisms (SNPs). Using a combined phylogenetic and species delimitation approach we uncovered two distinct species, as well as a well-defined third group that may contain multiple species. The findings are discussed with respect to possible drivers of diversification and the epidemiological importance of the distinct species and groups.

Protein mass spectrometry extends temporal blood meal detection over polymerase chain reaction in mouse-fed Chagas disease vectors

BACKGROUND Chagas disease is highly prevalent in Latin America, and vector control is the most effective control strategy to date. We have previously shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) is a valuable tool for identifying triatomine vector blood meals. OBJECTIVES The purpose of this study was to determine blood meal detection ability as a function of method [polymerase chain reaction (PCR) vs. LC-MS/MS], time since feeding, and the effect of molting in mouse-fed triatomine insect vectors targeting hemoglobin and albumin proteins with LC-MS/MS and short interspersed nuclear elements (SINE)-based PCR. METHODS We experimentally fed Triatoma protracta on mice and used LC-MS/MS to detect hemoglobin and albumin peptides over time post-feeding and post-molting (≤ 12 weeks). We compared LC-MS/MS results with those of a standard PCR method based on SINEs. FINDINGS Hemoglobin-based LC-MS/MS detected blood meals most robustly at all time points post-feeding. Post-molting, no blood meals were detected with PCR, whereas LC-MS/MS detected mouse hemoglobin and albumin up to 12 weeks. MAIN CONCLUSIONS In our study, the hemoglobin signature in the insect abdomen lasted longer than that of albumin and DNA. LC-MS/MS using hemoglobin shows promise for identifying triatomine blood meals over long temporal scales and even post-molting. Clarifying the frequency of blood-feeding on different hosts can foster our understanding of vector behavior and may help devise sounder disease-control strategies, including Ecohealth (community based ecosystem management) approaches.

Chagas disease vector blood meal sources identified by protein mass spectrometry.

Chagas disease is a complex vector borne parasitic disease involving blood feeding Triatominae (Hemiptera: Reduviidae) insects, also known as kissing bugs, and the vertebrates they feed on. This disease has tremendous impacts on millions of people and is a global health problem. The etiological agent of Chagas disease, Trypanosoma cruzi (Kinetoplastea: Trypanosomatida: Trypanosomatidae), is deposited on the mammalian host in the insect's feces during a blood meal, and enters the host's blood stream through mucous membranes or a break in the skin. Identifying the blood meal sources of triatomine vectors is critical in understanding Chagas disease transmission dynamics, can lead to identification of other vertebrates important in the transmission cycle, and aids management decisions. The latter is particularly important as there is little in the way of effective therapeutics for Chagas disease. Several techniques, mostly DNA-based, are available for blood meal identification. However, further methods are needed, particularly when sample conditions lead to low-quality DNA or to assess the risk of human cross-contamination. We demonstrate a proteomics-based approach, using liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify host-specific hemoglobin peptides for blood meal identification in mouse blood control samples and apply LC-MS/MS for the first time to Triatoma dimidiata insect vectors, tracing blood sources to species. In contrast to most proteins, hemoglobin, stabilized by iron, is incredibly stable even being preserved through geologic time. We compared blood stored with and without an anticoagulant and examined field-collected insect specimens stored in suboptimal conditions such as at room temperature for long periods of time. To our knowledge, this is the first study using LC-MS/MS on field-collected arthropod disease vectors to identify blood meal composition, and where blood meal identification was confirmed with more traditional DNA-based methods. We also demonstrate the potential of synthetic peptide standards to estimate relative amounts of hemoglobin acquired when insects feed on multiple blood sources. These LC-MS/MS methods can contribute to developing Ecohealth control strategies for Chagas disease transmission and can be applied to other arthropod disease vectors.

The diversity of the Chagas parasite, Trypanosoma cruzi, infecting the main Central American vector, Triatoma dimidiata, from Mexico to Colombia.

Little is known about the strains of Trypanosoma cruzi circulating in Central America and specifically in the most important vector in this region, Triatoma dimidiata. Approximately six million people are infected with T. cruzi, the causative agent of Chagas disease, which has the greatest negative economic impact and is responsible for ~12,000 deaths annually in Latin America. By international consensus, strains of T. cruzi are divided into six monophyletic clades called discrete typing units (DTUs TcI-VI) and a seventh DTU first identified in bats called TcBat. TcI shows the greatest geographic range and diversity. Identifying strains present and diversity within these strains is important as different strains and their genotypes may cause different pathologies and may circulate in different localities and transmission cycles, thus impacting control efforts, treatment and vaccine development. To determine parasite strains present in T. dimidiata across its geographic range from Mexico to Colombia, we isolated abdominal DNA from T. dimidiata and determined which specimens were infected with T. cruzi by PCR. Strains from infected insects were determined by comparing the sequence of the 18S rDNA and the spliced-leader intergenic region to typed strains in GenBank. Two DTUs were found: 94% of infected T. dimidiata contained TcI and 6% contained TcIV. TcI exhibited high genetic diversity. Geographic structure of TcI haplotypes was evident by Principal Component and Median-Joining Network analyses as well as a significant result in the Mantel test, indicating isolation by distance. There was little evidence of association with TcI haplotypes and host/vector or ecotope. This study provides new information about the strains circulating in the most important Chagas vector in Central America and reveals considerable variability within TcI as well as geographic structuring at this large geographic scale. The lack of association with particular vectors/hosts or ecotopes suggests the parasites are moving among vectors/hosts and ecotopes therefore a comprehensive approach, such as the Ecohealth approach that makes houses refractory to the vectors will be needed to successfully halt transmission of Chagas disease.