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Lake Tzibaná is one of the largest (1.27 km2) and deepest (Zmax = 52 m) karstic lakes in the UNESCO’s Biosphere Reserve “Nahá-Metzabok” and in the Lacandon Forest, southeastern Mexico. It archives sediments from multiple sources and the inflowing Nahá River forms deltaic deposits. In 2019, the water level in Lake Tzibaná declined by ~ 15 m, persisting for 4 months and exposing the Nahá River Delta. A geophysical profile on the exposed delta revealed an accumulation of ~ 20 m of such deposits. Three sediment outcrops from an inactive channel in the Nahá River Delta, which ranged in height from 0.6 to 1.43 m, were sampled and a multi-proxy analysis of biological remains and geochemical variables was conducted. Four facies were observed: (1) massive-coarse sand, (2) fine sand, (3) dark leaf litter and (4) massive silty clay, each characterized by specific microcrustacean, testate amoebae and diatom taxa. Six dark leaf litter horizons were radiocarbon dated and revealed a complex depositional history including inverted ages making the establishment of an age model difficult. Nevertheless, past lake-level changes and the formation of the four facies match three characteristic water-level stages, which can also be observed on recent satellite images: (1) Massive-coarse sand deposits, with compositional and sedimentological characteristics of a shoreline environment and fluvial lateral banks, were formed during large-magnitude reductions in the lake level, similar to the one in 2019, (2) Interbedded layers of fine sand and dark leaf litter, currently found in low-energy fluvial environments, were formed during shorter and less pronounced decrease intervals, and (3) Massive silty clay, with distinctive microorganisms from low-energy lacustrine environments, is deposited during high water-level stages, when the delta is covered by water. Our findings illustrate how hydrological changes alter sedimentary dynamics in deltaic areas of lakes. Despite the complexity of their depositional processes, deltaic records can serve as a complementary source of paleolimnological information to records from distal zones due to their sensitivity to variations in water level, especially during extreme and prolonged desiccation events. Future research should attempt to combine evidences from deltaic and sediment sequences from deeper zones of Lake Tzibaná to reconstruct water-level variations during the entire Holocene. Understanding past lake-level reductions is not only relevant for the local indigenous communities but also crucial for the conservation of this ecosystem of international importance.

Metagenomic and traditional paleolimnological approaches are suitable to infer past biological and environmental changes, however, they are often applied independently, especially in tropical regions. We combined both approaches to investigate Holocene Prokaryote and Eukaryote diversity and microbial metabolic pathways in ancient Lake Chalco, Mexico. Here, we report on diversity among a large number of lineages (36,722 OTUs) and functional diversity (27,636,243 non-clustered predicted proteins, and 6,144 annotated protein-family genes). The most abundant domain is Bacteria (81%), followed by Archaea (15%) and Eukarya (3%). We also determined the diversity of protein families and their relationship to metabolic pathways. The early Holocene (> 11,000 cal years BP) lake was characterized by cool, freshwater conditions, which later became warmer and hyposaline (11,000–6,000 cal years BP). We found high abundances of cyanobacteria, and fungi groups associated with mature forests in these sediments. Bacteria and Archaea include mainly anaerobes and extremophiles that are involved in the sulfur, nitrogen, and carbon cycles. We found evidence for early human impacts, including landscape modifications and lake eutrophication, which began ~ 6,000 cal years BP. Subsaline, temperate conditions were inferred for the past 5,000 years. Finally, we found nitrogen-fixing bacteria and protein-family genes that are linked to contaminated environments, as well as several fungal pathogens of crops in near-surface sediments.

For decades, paleoecological studies in lake sediments have focused on reconstructing the environments of the past and explaining phenomena linked to climatic variations. Recent advances in high-throughput DNA sequencing have allowed access to environmental DNA (eDNA) and ancient sedimentary DNA (sedaDNA) as a new and efficient proxy for past and present biodiversity. The basin of Mexico (BM) is located in the central part of the Trans-Mexican Volcanic Belt at 2,200 m a.s.l.; with the southern portion harboring the Chalco sub-basin. Lake Chalco is one of the last remaining natural aquatic ecosystems within the ever-expanding urban area surrounding Mexico City. The paleoenvironmental history of this lake has been previously characterized using sedimentological and geochemical proxies, as well as preserved microfossils (diatoms, pollen) with a temporal framework based on multiple radiocarbon dates. However, information for the remaining taxonomic groups and metabolic pathways remained unexplored. Here, we present the first metagenomics-based study for the Holocene in a high-altitude lake in Central Mexico –Lake Chalco. We explored the relationship between the lake’s paleoenvironmental condition and estimations of taxonomic and metabolic profiles across the sedimentary sequence (2.5 meters long). Multiple biological and abiotic variables revealed three main environmental phases: 1) a cool freshwater lake (FW1: 11,500-11,000 cal years BP), 2) a warm hyposaline lake (HS2: 11,000-6,000 cal years BP), and 3) a temperate, subsaline lake (SS3

Detailed knowledge of species ecological preferences and robust taxonomy of paleobioindicators are prerequisites for accurate paleoclimate and paleoenvironmental studies. This study aims to expand the knowledge of modern, Neotropical freshwater ostracode fauna, across an altitudinal gradient from the karst lakes in the lowlands of El Petén, Guatemala (100-500 m.a.s.l.), to the mid-elevation lakes of the Lacandón forest (500-1 000 m.a.s.l.), to the higher-altitude lakes of Montebello, Chiapas, Mexico (1 000-1 500 m.a.s.l.). Eighteen ostracode species were identified in 24 lakes. Ostracodes were absent in Lakes Amarillo and Lacandón (mid-altitude), and San Diego (lowlands); probably explained by a structural difference of habitats and species interactions. Statistical analysis indicated that the most abundant species, Cypridopsis vidua (O.F. Müller, 1776), Cytheridella ilosvayi (Daday, 1905), Pseudocandona antillana (Broodbakker, 1983), and Darwinula stevensoni (Brady & Robertson, 1870) have a continuous distribution along the entire altitudinal gradient. Some species display more restricted distributions, determined by temperature, precipitation and conductivity. For example, Eucypris sp. is restricted to the lowlands, Vestalenula sp. and Cypria sp. were found only at middle elevations. Species diversity is slightly greater in lakes at middle altitudes (Haverage = 1.09) than in water bodies in the lowlands (Haverage = 0.94) and in cooler lakes in the highlands (Haverage = 0.94), suggesting that mid-elevation lakes have a high potential for harboring microrefugia. Locally weighted scatterplot smoothing (LOESS regressions) provided ecological preference information for the four most frequent and widely distributed species, with respect to temperature, conductivity, bicarbonate (HCO3 -) concentration, precipitation, and pH. Darwinula stevensoni suggest an association more to cooler temperatures and lower conductivities proving its high tolerance range. Cypridopsis vidua is associated with warm and low-rainfall environments, such as recorded in the lowlands of Guatemala, and can be used as a paleobioindicator of vegetated littoral zones, because we found it always associated to this section of lakes. Cytheridella ilosvayi show preferences for warm and humid conditions, whereas P. antillana prefer colder and humid environments. Such quantitative-ecological information will improve ostracode-based paleoenvironmental reconstructions in Southern Mexico and Northern Guatemala. In addition, our approach serves as a model for future paleoecological studies that employ other aquatic bioindicators, such as testate amoebae, cladocerans, and chironomids. RESUMEN Los ostrácodos son microcrustáceos acuáticos, que poseen un caparazón bivalvo de carbonato de calcio que se puede preservar en los sedimentos de los ambientes lacustres. Debido a su alta sensibilidad a cambios ambientales y su alta potencialidad de fosilización, los ostrácodos son una herramienta útil para el estudio paleoclimático y paleoambiental, abarcando una temporalidad de décadas hasta millones de años. El conocimiento ecológico de las especies, así como su taxonomía son prerrequisitos para estos estudios. Sin embargo, esta información es aún escasa en diferentes regiones del mundo, incluyendo los Neotrópicos. Hasta el momento, se han realizado únicamente estudios en los lagos kársticos de las tierras bajas de la Península de Yucatán, en el norte del Neotrópico. Sin embargo, los lagos en altitudes medias y altas permanecen poco conocidos. El objetivo de este trabajo es aportar conocimiento de ostrácodos no-marinos neotropicales a lo largo de un gradiente altitudinal que va desde las tierras bajas de El Petén, Guatemala (100-500 m s.n.m.), incluyendo los lagos de tierras medias de la Selva Lacandona (500-1 000 m s.n.m.), hasta las tierras altas de Montebello, Chiapas, México (1 000-1 500 m s.n.m.). Dieciocho especies de ostrácodos se identificaron en 24 lagos, pero estuvieron ausentes en los lagos Amarillo y Lacandón (tierras medias) y San Diego (tierras bajas). La ausencia de ostrácodos podría explicarse por la falta de un muestreo estacional o por variables que no se consideraron en este estudio como sustrato, cobertura vegetal acuática e interacciones interespecíficas. Los análisis estadísticos indicaron que las especies más abundantes son: Cypridopsis vidua (O.F. Müller, 1776), Cytheridella ilosvayi (Daday, 1905), Pseudocandona antillana (Broodbakker, 1983) y Darwinula stevensoni (Brady & Robertson, 1870), con una distribución continua a lo largo del gradiente altitudinal. Algunas especies presentan una distribución más restringida, determinada por la temperatura, precipitación y conductividad. Por ejemplo, Eucypris sp. está restringida a las tierras bajas; mientras que Vestalenula sp. y Cypria sp. se encontraron únicamente en elevaciones medias. La diversidad de especies es ligeramente mayor en lagos cálidos a altitudes medias (Haverage = 1.09) que en las tierras bajas (Haverage = 0.94) y que en lagos de agua más fría en las tierras altas (Haverage = 0.94), sugiriendo que los lagos de tierras medias tienen un alto potencial para albergar micro-refugios. Las regresiones LOESS muestran las preferencias ecológicas de las cuatro especies más frecuentes y altamente distribuidas con respecto a la temperatura, conductividad, HCO3 -, precipitación y pH. Darwinula stevensoni se asocia a temperaturas frías y conductividades bajas, lo que evidencia su alto rango de tolerancia. Cypridopsis vidua se asocia con ambientes cálidos y de baja precipitación, como los registrados en las tierras bajas de Guatemala, y puede usarse como paleobioindicador de zonas de vegetación litoral. Cytheridella ilosvayi es un indicador de condiciones cálidas y húmedas, mientras que P. antillana de frías y húmedas. Esta información ecológica-cuantitativa se podrá utilizar como una herramienta para las reconstrucciones paleoambientales basadas en ostrácodos en el sur de México y norte de Guatemala. Además, este enfoque sirve como modelo para futuros estudios paleoecológicos que emplean otros bioindicadores acuáticos, como las amebas testadas, los cladóceros y los quironómidos.

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). The protein extracts of T. solium cysts are complex mixtures including cyst's and host proteins. Little is known about the influence of using different detergents in the efficiency of solubilization-extraction of these proteins, including relevant antigens. Here, we describe the use of CHAPS, ASB-14 and Triton X-100, alone or in combination in the extraction buffers, as a strategy to notably increase the recovery of proteins that are usually left aside in insoluble fractions of cysts. Using buffer with CHAPS alone, 315 protein spots were detected through 2D-PAGE. A total of 255 and 258 spots were detected using buffers with Triton X-100 or ASB-14, respectively. More protein spots were detected when detergents were combined, i.e., 2% CHAPS, 1% Triton X-100 and 1% ASB-14 allowed detection of up to 368 spots. Our results indicated that insoluble fractions of T. solium cysts were rich in antigens, including several glycoproteins that were sensitive to metaperiodate treatment. Host proteins, a common component in protein extracts of cysts, were present in larger amounts in soluble than insoluble fractions of cysts proteins. Finally, antigens present in the insoluble fraction were more appropriate as a source of antigens for diagnostic procedures.

Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

The recommended chemotherapy for drug-sensitive tuberculosis (TB) consists of four different antibiotics administrated for 6 months. This long treatment leads to significant compliance problems and consequently to recrudescence of the disease and to the development of multidrug-resistant (MDR) strains. Thus, new alternatives are needed to shorten or simplify the treatment of TB. Antibodies have therapeutic effects in animal models of TB, so their use as adjuvants in drug-sensitive and MDR TB is an interesting alternative. To assess the effect of antibodies, BALB/c mice with active late disease 60 days after infection with drug-sensitive TB strain H37Rv were treated with intravenous immunoglobulin (IVIg), alone or in combination with conventional chemotherapy. When compared with control non-treated animals, IVIg alone produced a significantly decreased burden of pulmonary bacilli. This decrease was even greater when IVIg was used in combination with conventional chemotherapy. The combined therapy also significantly reduced tissue damage (pneumonia) when compared to infected animals treated only with antibiotics. IVIg treatment also caused decreased bacillary burdens in mice infected with an MDR strain. In vitro experiments suggested that improving phagocytosis by efficient opsonization is perhaps the principal mechanism of this beneficial therapeutic effect.

The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition, which allows the parasite to survive temporarily inside a chitin-like resistant cover containing Jacob protein. Our findings lead us to suggest that encystment and CLS formation could be distinct stress responses. In addition, we show that cysts express a high number of genes with unknown function, including four new, highly antigenic, possibly membrane-located proteins that could be targets of therapeutic and diagnostic usefulness.

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). Infestation of the human brain, also known as neurocysticercosis, is the most common parasite disease of the central nervous system worldwide. Significant advances in the understanding of the disease have been achieved using the Taenia crassiceps murine model. We describe here a successful transfection protocol of T. crassiceps cysticerci as the first step to approach a number of currently inaccessible biological questions on cysticercosis. T. crassiceps cysticerci (ORF strain) were microinjected with the plasmid pcDNA3.1/NT-GFP-TOPO, encoding the green fluorescent protein (GFP) driven by a cytomegalovirus promoter (CMV). Twelve hours after the microinjection, GFP fluorescence gradually developed in patches associated to bud structures in the bladder wall of cysts. Fluorescence reached a peak at 24–48 h and lasted up to 72 h after the microinjection. Immunohistochemical studies on tissue sections of transfected cysts using an anti-GFP antibody, demonstrated co-localization of the antibody and the GFP fluorescence in the tegumentary cytoplasm and subtegumentary cytons. To validate at the mRNA level the expression of GFP, we carried out RT-PCR using two pairs of nested primers. Results showed expression of GFP-mRNA at 24 h post-transfection. Moreover, western blot assays of crude extracts of transfected cysts, carried out using the anti-GFP specific antibody, showed the expected protein band of 27 kDa, demonstrating that the GFP expression started at 24 after plasmid microinjection and was maintained up to 72 h. These findings will facilitate the development of functional genomics approaches applied to this model of cysticercosis. Electronic supplementary material The online version of this article (doi:10.1186/s40064-015-1278-y) contains supplementary material, which is available to authorized users.

The host–parasite relationship in cestode infections is complex. One feature of this bidirectional molecular communication is the uptake of host proteins by the parasite. Here we describe the presence of several host proteins in the vesicular fluid of Taenia solium cysticerci dissected from the central nervous system and the skeletal muscle of naturally infected pigs. Using two-dimensional electrophoresis we compared the protein patterns of vesicular fluids of cysticerci vs. the sera of cysticercotic pigs. We found that the vesicular fluids of both groups of cysts showed 17 protein spots matching with the pig’s sera spots. After mass spectrometry sequencing of these spots, five host proteins were identified: hemoglobin, albumin, serpin A3–8, haptoglobin, rho GTPase-activating protein 36-like. Three of the 17 spots corresponded to host protein fragments: hemoglobin, albumin and serpin A3–8. IgG heavy and light chains were also identified by Western blot using a specific antibody. Quantitative estimations indicated that the host proteins represented 11–13% of the protein content in the vesicular fluids. We also calculated the relative abundance of these host proteins in the vesicular fluids; all were represented in similar relative abundances as in host sera. This suggests that uptake of host proteins by cysticerci proceeds through an unspecific mechanism such as non-specific fluid pinocytosis.

En Amerique latine, plus de 10 millions de personnes sont porteuses d’un parasite qui les rend susceptibles de developper la maladie de Chagas. Cette maladie chronique et invalidante est causee par un parasite hematophage microscopique, appele Trypanosoma cruzi. La transmission du parasite est attribuable a plusieurs especes d’insectes, dont les punaises de lit ou triatomes. En general, cette maladie se contracte pendant l’enfance et presente des symptomes pseudogrippaux. Toutefois, des symptomes plus severes touchant le systeme digestif et le cœur apparaissent plus tard chez un tiers environ des personnes atteintes. La maladie de Chagas tue plus de 10 000 personnes par an (OMS 2013).

In Latin America, more than 10 million people carry a parasite that puts them at risk of developing Chagas disease. This chronic and debilitating illness is caused by a microscopic blood parasite called Trypanosoma cruzi.

The deterioration or absence of plaster walls in houses and poor hygienic conditions are the most important risk factors for indoor Triatoma dimidiata infestation in Guatemala. A cross-disciplinary study was conducted addressing T. dimidiata infestation, household hygiene, and housing construction. The study focused on local materials and cultural aspects (including gender roles) that could lead to long-term improvements in wall construction. A new plaster mix for walls was developed on the basis of laboratory studies on construction materials recommended by local villagers. Four villages with persistent (post-spraying) T. dimidiata infestation were studied. In two villages, an ecosystem approach was implemented, and the homeowners conducted wall improvements and household sanitation with the support of the interdisciplinary team (the ecosystem intervention). In the other two villages, a vector control approach based on insecticide spraying was adopted (traditional intervention). Both interventions were associated with a reduction in T. dimidiata infestation, but only the ecosystem approach produced important housing improvements (sanitation and wall construction) capable of preventing T. dimidiata re-infestation in the long term. A degradação ou ausência de reboco nas paredes e as condições higiênicas deficientes são os fatores de risco mais importantes para a infestação intradomiciliar por Triatoma dimidiata na Guatemala. Realizamos um estudo transdisciplinar sobre infestação por T. dimidiata, higiene intradomiciliar e condições de construção. O estudo destacou as questões de materiais locais e aspectos culturais (inclusive papéis de gênero) que poderiam levar a melhorias nas condições das paredes no longo prazo. Formulou-se uma nova mistura de gesso para paredes, após estudos de laboratório sobre materiais de construção com base em recomendações dos residentes locais. Foram estudados quatro vilarejos com infestação por T. dimidiata que havia persistido mesmo após aplicação de inseticida. Em duas comunidades, foi implementada uma abordagem ecossistêmica, e os residentes implementaram melhorias nas paredes e no saneamento domiciliar, com o apoio da equipe interdisciplinar (intervenção ecossistêmica). Nas duas outras comunidades, adotou-se uma estratégia de controle com base na aplicação de inseticida (intervenção tradicional). Ambas as intervenções levaram a uma redução na infestação por T. dimidiata, mas apenas a abordagem ecossistêmica produziu melhorias importantes nas condições de moradia (saneamento e revestimento das paredes) capazes de prevenir a re-infestação por T. dimidiata no longo prazo.

Seventeen variables were evaluated as possible risk factors for the intradomiciliary infestation with Triatoma dimidiata in 644 houses in Jutiapa, Guatemala. During 2004 the houses were assessed for vector presence and evaluated for hygiene, cluttering, material comfort, construction conditions and number of inhabitants, among other factors. Chi-square analysis detected significant associations between vector presence and eight variables related to domestic sanitary and construction conditions. Log-linear models showed that regardless of the age of the house, the odds of vector presence were 4.3 and 10 times lower in houses with a good socioeconomic status compared with poor and very poor houses respectively. Log-linear models also pointed to a greater chance of vector presence when walls lacked plastering (3.85 times) or walls had low quality-incomplete plastering (4.56 times), compared with walls that were completely plastered. Control strategies against T. dimidiata should include the introduction of better-quality but inexpensive plastering formulations and better sanitation practices should also be promoted among the population. Such control strategies should not only reduce or eliminate infestation, but also prevent vector reinfestation. Seiscientas cuarenta y cuatro casas en Jutiapa, Guatemala fueron encuestadas en el año 2004 para buscar el vector de la enfermedad de Chagas Triatoma dimidiata. Diecisiete variables relacionadas con las condiciones estructurales y de higiene de las casas fueron registradas y evaluadas como factores de riesgo para la infestación intradomiciliar con T. dimidiata. Análisis chi-cuadrado detectaron asociaciones significativas entre la presencia del vector y 8 de estas variables. En modelos log-lineares se detectó, que sin importar la antigüedad de la casa, las posibilidades de presencia del vector fueron 4,3 y 10 veces más bajas en casas con un buen estado socioeconómico que en casas pobres o muy pobres, respectivamente. Además, las posibilidades de infestación fueron mayores en casas con paredes sin repello (3,85 veces) o con repellado incompleto o de mala calidad (4,56 veces), que con repellado completo. Las estrategias para el control de T. dimidiata deberían incluir la introducción de repellos baratos y de buena calidad y la promoción de mejores prácticas sanitarias. Estas estrategias no solo reducirían o eliminaría la infestación, sino también ayudaría a prevenir la reinfestación.

Approximately 10 million people are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which remains the most serious parasitic disease in the Americas. Most people are infected via triatomine vectors. Transmission has been largely halted in South America in areas with predominantly domestic vectors. However, one of the main Chagas vectors in Mesoamerica, Triatoma dimidiata, poses special challenges to control due to its diversity across its large geographic range (from Mexico into northern South America), and peridomestic and sylvatic populations that repopulate houses following pesticide treatment. Recent evidence suggests T. dimidiata may be a complex of species, perhaps including cryptic species; taxonomic ambiguity which confounds control. The nuclear sequence of the internal transcribed spacer 2 (ITS2) of the ribosomal DNA and the mitochondrial cytochrome b (mt cyt b) gene were used to analyze the taxonomy of T. dimidiata from southern Mexico throughout Central America. ITS2 sequence divides T. dimidiata into four taxa. The first three are found mostly localized to specific geographic regions with some overlap: (1) southern Mexico and Guatemala (Group 2); (2) Guatemala, Honduras, El Salvador, Nicaragua, and Costa Rica (Group 1A); (3) and Panama (Group 1B). We extend ITS2 Group 1A south into Costa Rica, Group 2 into southern Guatemala and show the first information on isolates in Belize, identifying Groups 2 and 3 in that country. The fourth group (Group 3), a potential cryptic species, is dispersed across parts of Mexico, Guatemala, and Belize. We show it exists in sympatry with other groups in Peten, Guatemala, and Yucatan, Mexico. Mitochondrial cyt b data supports this putative cryptic species in sympatry with others. However, unlike the clear distinction of the remaining groups by ITS2, the remaining groups are not separated by mt cyt b. This work contributes to an understanding of the taxonomy and population subdivision of T. dimidiata, essential for designing effective control strategies., Author Summary The Chagas disease parasite, transmitted to humans by triatomine bugs, remains a leading cause of heart and digestive disease in Latin America. Pesticide spraying has effectively halted transmission in most of southern South America, especially where the bugs live exclusively inside houses. In Mesoamerica, bugs living in the forest readily reinfest treated houses. In addition, one of the main species of insect that transmits Chagas in Mesoamerica, Triatoma dimidiata, although it looks similar in different localities, may consist of genetically distinct populations, even different species, which differ in how efficiently they transmit the parasite: characteristics which confound control efforts. Nuclear and mitochondrial DNA were analyzed to characterize different populations of T. dimidiata from Mexico and Central America. Both the nuclear and mitochondrial DNA show that there is a very distinct population of T. dimidiata, perhaps even a different species, that lives in very close proximity with other T. dimidiata in Mexico and Guatemala. The nuclear DNA divides the remaining T. dimidiata into three additional genetically distinct groups. However, the mitochondrial DNA does not distinguish these additional groups. This study helps inform control efforts by showing where genetically distinct populations of T. dimidiata occur.