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Background: A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. Results: The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq´s) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq´s between 24 and 33. Conclusions: This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles. Fil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Gómez Bravo, Andrea. Fundación Mundo Sano; Argentina Fil: Ramirez Gomez, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Pech May, Angélica del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Ramsey, Janine M.. Instituto Nacional de Salud Pública; México Fil: Abril, Marcelo. Fundación Mundo Sano; Argentina Fil: Guhl, Felipe. Universidad de los Andes; Colombia Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Vector-borne Chagas disease is endemic to the Americas and imposes significant economic and social burdens on public health. In a previous contribution, we presented an automated identification system that was able to discriminate among 12 Mexican and 39 Brazilian triatomine (Hemiptera: Reduviidae) species from digital images. To explore the same data more deeply using machine-learning approaches, hoping for improvements in classification, we employed TensorFlow, an open-source software platform for a deep learning algorithm. We trained the algorithm based on 405 images for Mexican triatomine species and 1,584 images for Brazilian triatomine species. Our system achieved 83.0 and 86.7% correct identification rates across all Mexican and Brazilian species, respectively, an improvement over comparable rates from statistical classifiers (80.3 and 83.9%, respectively). Incorporating distributional information to reduce numbers of species in analyses improved identification rates to 95.8% for Mexican species and 98.9% for Brazilian species. Given the ‘taxonomic impediment’ and difficulties in providing entomological expertise necessary to control such diseases, automating the identification process offers a potential partial solution to crucial challenges.

This study explores the present day distribution of Lutzomyia longipalpis in relation to climate, and transfers the knowledge gained to likely future climatic conditions to predict changes in the species' potential distribution. We used ecological niche models calibrated based on occurrences of the species complex from across its known geographic range. Anticipated distributional changes varied by region, from stability to expansion or decline. Overall, models indicated no significant north' south expansion beyond present boundaries. However, some areas suitable both at present and in the future (e.g., Pacific coast of Ecuador and Peru) may offer opportunities for distributional expansion. Our models anticipated potential range expansion in southern Brazil and Argentina, but were variably successful in anticipating specific cases. The most significant climate-related change anticipated in the species' range was with regard to range continuity in the Amazon Basin, which is likely to increase in coming decades. Rather than making detailed forecasts of actual locations where Lu. longipalpis will appear in coming years, our models make interesting and potentially important predictions of broader-scale distributional tendencies that can inform heath policy and mitigation efforts. Fil: Peterson, A. Townsend. University of Kansas. Biodiversity Research Center; Estados Unidos Fil: Campbell, Lindsay P.. University of Kansas. Biodiversity Research Center; Estados Unidos Fil: Moo Llanes, David A.. Instituto Nacional de Salud Pública; México Fil: Travi, Bruno. University of Texas; Estados Unidos Fil: González, Camila. Universidad de los Andes; Colombia Fil: Ferro, María Cristina. Instituto Nacional de Salud; Colombia Fil: Melim Ferreira, Gabriel Eduardo. Fundación Oswaldo Cruz; Brasil Fil: Brandão Filho, Sinval P.. Fundación Oswaldo Cruz; Brasil Fil: Cupolillo, Elisa. Fundación Oswaldo Cruz; Brasil Fil: Ramsey, Janine. Instituto Nacional de Salud Pública; México Fil: Leffer, Andreia Maruruto Chernaki. Universidade de Sao Paulo; Brasil Fil: Pech May, Angélica del Rosario. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Shaw, Jeffrey J.. Universidade de Sao Paulo; Brasil

Lutzomyia longipalpis is a complex of species which has a wide but discontinuous distribution from southeastern Mexico to northern Argentina and Uruguay. To date, eight mitochondrial haplogroups have been identified along its distribution although key environmental tolerances and ecological niche models have been analyzed only at the complex level. The aim of the present study was to analyze whether genetic diversification using three mitochondrial genes of the Lu. longipalpis complex is associated with niche divergence and to explore evolution of distributional projections of all haplogroups between the Last Glacial Maximum (LGM; 21,000 yrs ago) and the present. Current occurrence of all haplogroups was used to develop ecological niche models (ENM) and these were projected in both periods to quantify and identify geographic area shifts. Environmental space was used to estimate niche similarity between major clades and pairwise between individual haplogroups. The two major Lu. longipalpis clades (Mex, CA, Col and Ven vs Arg and Bra) had significantly different environmental space, indicating niche divergence. Environmental space overlap of southern haplogroups was variable, with divergent niche, except between Arg and ArgBra. The most suitable regions for the ArgBra haplogroup were northeastern and southeastern Brazil, and the Gran Chaco region. In contrast, ENM of haplogroups within the northern major clade have significantly similar niche, with highest geographic ENM suitability along both the Caribbean and Pacific coasts. The intensity and coverage of high suitability areas in the LGM decreased for most haplogroups in the present. Integrating ENM and phylogenetic analyses has allowed us to test hypotheses of niche similarity between Lu. longipalpis haplogroups and major clades, and to identify conserved distributional areas of haplogroups since the LGM, with the exception of Arg. Evidence for distributional shifts and overlap of haplogroups is important to analyze Leishmaniasis´ eco-epidemiology and to successfully monitor and control transmission. Fil: Moo Llanes, David A.. Instituto Nacional de Salud Pública. Centro Regional de Investigación en Salud Pública; México Fil: Pech May, Angélica del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Instituto Nacional de Salud Pública. Centro Regional de Investigación en Salud Pública; México. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Medicina Tropical.; Argentina Fil: Montes de Oca Aguilar, Ana Celia. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzados; México Fil: Salomón, Oscar Daniel. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Medicina Tropical.; Argentina. Universidad Nacional del Nordeste. Instituto de Medicina Regional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Ramsey, Janine. Instituto Nacional de Salud Pública. Centro Regional de Investigación en Salud Pública; México

The population genetics of Triatoma dimidiata haplogroups was analyzed at landscape and sub-regional scales in Chiapas and regional level across the Mexican Neotropics, and phylogeography of the complex was re-analyzed across its complete geographic range. Two contiguous fragments of the ND4 gene were analyzed due to bias from differential haplogroup specificity using a previously designed sequence. At both landscape (anthropic modification gradient) and regional (demographic, fragmentation, biogeographic, climate) scales, lowest T. dimidiata genetic diversity occurs where there is greatest historical anthropic modification, and where T. cruzi infection prevalence is significantly highest. Trypanosoma cruzi prevalence was significantly higher than expected in haplogroups 1 and 3, while lower than expected in haplogroup 2. There was also a significant difference of DTUI and DTUVI infection frequencies in both haplogroups 1 and 3, while no difference of either in haplogroup 2. All haplogroups from the Mexican Neotropics had moderate to high haplotype diversity, while greatest genetic differentiation was between haplogroups 1 and 3 (above FST = 0.868, p < 0.0001). Divergence of the complex from the MRCA was estimated between 0.97 MYA (95% HPD interval = 0.55–1.53 MYA) and 0.85 MYA (95% HPD interval = 0.42–1.5 MYA) for ND4A and both concatenated fragments, respectively, with primary divergence from the MRCA of haplogroups 2 and 3. Effective population size for Mexican haplogroups 1 and 2 increased between 0.02 and 0.03 MYA. This study supports previous ecological niche evidence for the complex´s origin surrounding the Tehuantepec Isthmus, and provides evidence for recent divergence of three primary dimidiata haplogroups, with differential T. cruzi infection frequency and DTU specificity, important components of vector capacity., Author summary Triatoma dimidiata is one of the broadest distributed triatomine species´ complexes transmitting Trypanosoma cruzi. In Mexico, three haplogroups of the T. dimidiata complex have been reported and all are primary vectors of Chagas disease south of the Tehuantepec Isthmus. Given their epidemiological importance, the question arises whether haplogroups have similar genetic diversity in domestic/modified landscapes, as well as infection characteristics and parasite DTU associations, key components of vector capacity. The aim of the present study was to analyze Triatoma dimidiata population genetics across landscapes, sub-regional, regional, and global Neotropical realm scales, using two contiguous fragments of the ND4 gene. Our results support previous evidence for the complex´s origin surrounding the Tehuantepec Isthmus, and provides evidence for recent divergence of three principal dimidiata haplogroups and significant secondary divergence within each. Differential T. cruzi prevalence and Discrete Typing Unit (DTU) specificity for individual haplogroups provide evidence for potential differential vector capacity within the complex in Mexico.

Background The Lutzomyia longipalpis complex has a wide but discontinuous distribution in Latin America, extending throughout the Neotropical realm between Mexico and northern Argentina and Uruguay. In the Americas, this sandfly is the main vector of Leishmania infantum, the parasite responsible for Visceral Leishmaniasis (VL). The Lu. longipalpis complex consists of at least four sibling species, however, there is no current consensus on the number of haplogroups, or on their divergence. Particularly in Argentina, there have been few genetic analyses of Lu. longipalpis, despite its southern expansion and recent colonization of urban environments. The aim of this study was to analyze the genetic diversity and structure of Lu. longipalpis from Argentina, and to integrate these data to re-evaluate the phylogeography of the Lu. longipalpis complex using mitochondrial markers at a Latin American scale. Methodology/Principal findings Genetic diversity was estimated from six sites in Argentina, using a fragment of the ND4 and the 3´ extreme of the cyt b genes. Greatest genetic diversity was found in Tartagal, Santo Tomé and San Ignacio. There was high genetic differentiation of Lu. longipalpis in Argentina using both markers: ND4 (FST = 0.452, p < 0.0001), cyt b (FST = 0.201, p < 0.0001). Genetic and spatial Geneland analyses reveal the existence of two primary genetic clusters in Argentina, cluster 1: Tartagal, Santo Tomé, and San Ignacio; cluster 2: Puerto Iguazú, Clorinda, and Corrientes city. Phylogeographic analyses using ND4 and cyt b gene sequences available in GenBank from diverse geographic sites suggest greater divergence than previously reported. At least eight haplogroups (three of these identified in Argentina), each separated by multiple mutational steps using the ND4, are differentiated across the Neotropical realm. The divergence of the Lu. longipalpis complex from its most recent common ancestor (MRCA) was estimated to have occurred 0.70 MYA (95% HPD interval = 0.48–0.99 MYA). Conclusions/Significance This study provides new evidence supporting two Lu. longipalpis genetic clusters and three of the total eight haplogroups circulating in Argentina. There was a high level of phylogeographic divergence among the eight haplogroups of the Lu. longipalpis complex across the Neotropical realm. These findings suggest the need to analyze vector competence, among other parameters intrinsic to a zoonosis, according to vector haplogroup, and to consider these in the design and surveillance of vector and transmission control strategies., Author summary The Lutzomyia longipalpis complex has a wide but discontinuous distribution in Latin America, extending throughout the Neotropical realm between Mexico and northern Argentina and Uruguay. In the Americas, this sandfly is the main vector of Leishmania infantum, the parasite responsible for Visceral Leishmaniasis (VL). The Lu. longipalpis complex is composed of at least four sibling species, although there is no current consensus on the number of haplogroups, or their divergence. In Argentina, little is known about the complex population structure. Therefore, the aim of this study was to analyze the diversity and genetic structure of Lu. longipalpis from Argentina and subsequently to analyze the complex phylogeography at a Latin American scale, using two mitochondrial markers. Greatest genetic diversity was found in Tartagal, Santo Tomé and San Ignacio. Two genetic clusters and three Lu. longipalpis haplogroups were identified from the six sites in Argentina. Phylogeographic analyzes using ND4 and cyt b gene sequences and those from across the Neotropical realm registered in GenBank, suggest greater divergence than previously reported. At least eight haplogroups are differentiated using the ND4, each separated by multiple mutational steps. Lu. longipalpis complex divergence from its most recent common ancestor (MRCA) was estimated at mid Pleistocene, 0.70 MYA (95% HPD interval = 0.48–0.99 MYA).

Landscape interactions of Trypanosoma cruzi (Tc) with Triatoma dimidiata (Td) depend on the presence and relative abundance of mammal hosts. This study analyzed a landscape adjacent to the Calakmul Biosphere Reserve, composed of conserved areas, crop and farming areas, and the human community of Zoh Laguna with reported Chagas disease cases. Sylvatic mammals of the Chiroptera, Rodentia, and Marsupialia orders were captured, and livestock and pets were sampled along with T. dimidiata in all habitats. Infection by T. cruzi was analyzed using mtDNA markers, while lineage and DTU was analyzed using the mini-exon. 303 sylvatic specimens were collected, corresponding to 19 species during the rainy season and 114 specimens of 18 species during dry season. Five bats Artibeus jamaicensis, Artibeus lituratus, Sturnira lilium, Sturnira ludovici, Dermanura phaeotis (Dp) and one rodent Heteromys gaumeri were collected in the three habitats. All but Dp, and including Carollia brevicauda and Myotis keaysi, were infected with predominately TcI in the sylvatic habitat and TcII in the ecotone. Sigmodon hispidus was the rodent with the highest prevalence of infection by T. cruzi I and II in ecotone and domestic habitats. Didelphis viginiana was infected only with TcI in both domestic and sylvatic habitats; the only two genotyped human cases were TcII. Two main clades of T. cruzi, lineages I (DTU Ia) and II (DTU VI), were found to be sympatric (all habitats and seasons) in the Zoh-Laguna landscape, suggesting that no species-specific interactions occur between the parasite and any mammal host, in any habitat. We have also found mixed infections of the two principal T. cruzi clades in individuals across modified habitats, particularly in livestock and pets, and in both haplogroups of T. dimidiata. Results are contradictory to the dilution hypothesis, although we did find that most resilient species had an important role as T. cruzi hosts. Our study detected some complex trends in parasite transmission related to lineage sorting within the matrix. Intriguingly, TcIa is dominant in terrestrial small wildlife in the sylvatic habitat and is the only parasite DTU found in D. virginiana in the domestic habitat, although its frequency remained constant in sylvatic and ecotone vectors. Bats have a key role in TcVI dispersal from the sylvatic habitat, while dogs, sheep, and humans are drivers of TcVI between domestic and ecotone habitats. Overall, our results allow us to conclude that T. cruzi transmission is dependent on host availability within a highly permeable landscape in Zoh Laguna. Fil: López Cancino, Sury Antonio. Instituto Nacional de Salud Pública; México Fil: Tun Ku, Ezequiel. Instituto Nacional de Salud Pública; México Fil: De la Cruz Felix, Himmler Keynes. Instituto Nacional de Salud Pública; México Fil: Ibarra Cerdena, Carlos Napoleón. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzados; México; Fil: Izeta Alberdi, Amaia. Instituto Nacional de Salud Publica; México Fil: Pech May, Angélica del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Instituto Nacional de Salud Publica; México Fil: Mazariegos Hidalgo, Carlos Jesús. Instituto Nacional de Salud Publica; México Fil: Valdez Tah, Alba Rocio. El Colegio de la Frontera Sur; México Fil: Ramsey, Janine. Instituto Nacional de Salud Publica; México

Chagas disease is one of the most important yet neglected parasitic diseases in Mexico and is transmitted by Triatominae. Nineteen of the 31 Mexican triatomine species have been consistently found to invade human houses and all have been found to be naturally infected with Trypanosoma cruzi. The present paper aims to produce a state-of-knowledge atlas of Mexican triatomines and analyse their geographic associations with T. cruzi, human demographics and landscape modification. Ecological niche models (ENMs) were constructed for the 19 species with more than 10 records in North America, as well as for T. cruzi. The 2010 Mexican national census and the 2007 National Forestry Inventory were used to analyse overlap patterns with ENMs. Niche breadth was greatest in species from the semiarid Nearctic Region, whereas species richness was associated with topographic heterogeneity in the Neotropical Region, particularly along the Pacific Coast. Three species, Triatoma longipennis, Triatoma mexicana and Triatoma barberi, overlapped with the greatest numbers of human communities, but these communities had the lowest rural/urban population ratios. Triatomine vectors have urbanised in most regions, demonstrating a high tolerance to human-modified habitats and broadened historical ranges, exposing more than 88% of the Mexican population and leaving few areas in Mexico without the potential for T. cruzi transmission.

Background The Triatoma phyllosoma complex of Trypanosoma cruzi vectors (Triatominae: Reduviidae) is distributed in both Neotropical and Nearctic bioregions of Mexico. Methods Volatile organic compounds emitted by disturbed Triatoma longipennis, Triatoma pallidipennis and Triatoma phyllosoma, and from their Brindley’s and metasternal glands, were identified using solid-phase microextraction coupled with gas chromatography-mass spectrometry. Results Disturbed bugs and the metasternal glands from T. phyllosoma released or had significantly fewer compounds than T. longipennis and T. pallidipennis. Isobutyric acid was the most abundant compound secreted by disturbed bugs of the three species, while Brindley’s glands of all species produced another four compounds: propanoic acid, isobutyric acid, pentyl butanoate, and 2-methyl hexanoic acid. Two novel compounds, both rose oxide isomers, were produced in MGs and released only by disturbed females of all three species, making this the first report in Triatominae of these monoterpenes. The principal compound in MGs of both sexes of T. longipennis and T. phyllosoma was 3-methyl-2-hexanone, while cis-rose oxide was the principal compound in T. pallidipennis females. The major components in male effluvia of T. pallidipennis were 2-decanol and 3-methyl-2-hexanone. Conclusion Discriminant analysis of volatile organic compounds was significant, separating the three species and was consistent with morphological and genetic evidence for species distinctions within the complex.

Nine sandfly species (Diptera: Psychodidae) are suspected or proven vectors of Leishmania spp. in the North and Central America region. The ecological niches for these nine species were modelled in three time periods and the overlaps for all time periods of the geographic predictions (G space), and of ecological dimensions using pairwise comparisons of equivalent niches (E space), were calculated. Two Nearctic, six Neotropical and one species in both bioregions occupied a reduced number of distribution areas. The ecological niche projections for most sandfly species other than Lutzomyia shannoni and Lutzomyia ovallesi have not expanded significantly since the Pleistocene. Only three species increase significantly to 2050, whereas all others remain stable. Lutzomyia longipalpis shared a similar ecological niche with more species than any other, although both L. longipalpis and Lutzomyia olmeca olmeca had conserved distributions over time. Climate change, at both regional and local levels, will play a significant role in the temporal and spatial distributions of sandfly species. Fil: Moo Llanes, David A.. Universidad Nacional Autónoma de México; México Fil: Pech May, Angélica del Rosario. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Ibarra Cerdeña, C. N.. Cinvestav Unidad Merida; México. Universidad Nacional Autónoma de México; México Fil: Rebollar Téllez, E. A.. Universidad Autónoma de Nuevo León; México Fil: Ramsey, Janine. Instituto Nacional de Salud Pública/crisp; México

Background The evolutionary history and ecological associations of Trypanosoma cruzi, the need to identify genetic markers that can distinguish parasite subpopulations, and understanding the parasite’s evolutionary and selective processes have been the subject of a significant number of publications since 1998, the year when the first DNA sequence analysis for the species was published. Methods The current analysis systematizes and re-analyzes this original research, focusing on critical methodological and analytical variables and results that have given rise to interpretations of putative patterns of genetic diversity and diversification of T. cruzi lineages, discrete typing units (DTUs), and populations, and their associations with hosts, vectors, and geographical distribution that have been interpreted as evidence for parasite subpopulation specificities. Results Few studies use hypothesis-driven or quantitative analysis for T. cruzi phylogeny (16/58 studies) or phylogeography (10/13). Among these, only one phylogenetic and five phylogeographic studies analyzed molecular markers directly from tissues (i.e. not from isolates). Analysis of T. cruzi DTU or lineage niche and its geographical projection demonstrate extensive sympatry among all clades across the continent and no significant niche differences among DTUs. DTU beta-diversity was high, indicating diverse host assemblages across regions, while host dissimilarity was principally due to host species turnover and to a much lesser degree to nestedness. DTU-host order specificities appear related to trophic or microenvironmental interactions. Conclusions More rigorous study designs and analyses will be required to discern evolutionary processes and the impact of landscape modification on population dynamics and risk for T. cruzi transmission to humans. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1918-2) contains supplementary material, which is available to authorized users.

In this study, we evaluated the responses of Triatoma dimidiata Latreille (Hemiptera: Reduviidae) to volatiles emitted by conspecific females, males, mating pairs and metasternal gland (MG) extracts with a Y-tube olfactometer. The volatile compounds released by mating pairs and MGs of T. dimidiata were identified using solid-phase microextraction and coupled gas chromatography-mass spectrometry (GC-MS). Females were not attracted to volatiles emitted by males or MG extracts; however, they preferred clean air to their own volatiles or those from mating pairs. Males were attracted to volatiles emitted by males, females, mating pairs, pairs in which the male had the MG orifices occluded or MG extracts of both sexes. However, males were not attracted to volatiles emitted by pairs in which the female had the MG orifices occluded. The chemical analyses showed that 14 and 15 compounds were detected in the headspace of mating pairs and MG, respectively. Most of the compounds identified from MG except for isobutyric acid were also detected in the headspace of mating pairs. Both females and males were attracted to octanal and 6-methyl-5-hepten-2-one, and males were attracted to 3,5-dimethyl-2-hexanol. Males but not females were attracted to a seven-compound blend, formulated from compounds identified in attractive MG extracts.

Development of new genetic approaches to either interfere with the ability of mosquitoes to transmit dengue virus or to reduce vector population density requires progressive evaluation from the laboratory to contained field trials, before open field release. Trials in contained outdoor facilities are an important part of this process because they can be used to evaluate the effectiveness and reliability of modified strains in settings that include natural environmental variations without releasing mosquitoes into the open field. We describe a simple and cost-effective semi-field system designed to study Aedes aegypti carrying a dominant lethal gene (fsRIDL) in semi-field conditions. We provide a protocol for establishing, maintaining, and monitoring stable Ae. aegypti population densities inside field cages.

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

Triatoma dimidiata (Latreille) is a species complex that spans North, Central, and South America and which is a key vector of all known discrete typing units (DTU) of Trypanosoma cruzi, the etiologic agent of Chagas disease. Morphological and genetic studies indicate that T. dimidiata is a species complex with three principal haplogroups (hg) in Mexico. Different markers and traits are still inconclusive regarding if other morphological differentiation may indicate probable behavioral and vectorial divergences within this complex. In this paper we compared the antennae of three Mexican haplogroups (previously verified by molecular markers ND4 and ITS-2) and discussed possible relationships with their capacity to disperse and colonized new habitats. The abundance of each type of sensillum (bristles, basiconics, thick- and thin-walled trichoids) on the antennae of the three haplogroups, were measured under light microscopy and compared using Kruskal-Wallis non-parametric and multivariate non-parametric analyses. Discriminant analyses indicate significant differences among the antennal phenotype of haplogroups either for adults and some nymphal stages, indicating consistency of the character to analyze intraspecific variability within the complex. The present study shows that the adult antennal pedicel of the T. dimidiata complex have abundant chemosensory sensilla, according with good capacity for dispersal and invasion of different habitats also related to their high capacity to adapt to conserved as well as modified habitats. However, the numerical differences among the haplogroups are suggesting variations in that capacity. The results here presented support the evidence of T. dimidiata as a species complex but show females and males in a different way. Given the close link between the bug's sensory system and its habitat and host-seeking behavior, AP characterization could be useful to complement genetic, neurological and ethological studies of the closely related Dimidiata Complex haplogroups for a better knowledge of their vectorial capacity and a more robust species differentiation. Fil: May Concha, Irving Jesus. Provincia de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Universidad Autónoma de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción; Argentina. Centro Regional de Investigación en Salud Pública; México Fil: Guerenstein, Pablo Gustavo. Provincia de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Universidad Autónoma de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción; Argentina Fil: Ramsey, Janine. Centro Regional de Investigación en Salud Pública; México Fil: Rojas, Julio C.. Colegio de la Frontera Sur; México Fil: Catala, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja. - Universidad Nacional de La Rioja. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja. - Universidad Nacional de Catamarca. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja. - Secretaría de Industria y Minería. Servicio Geológico Minero Argentino. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja. - Provincia de La Rioja. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja; Argentina

The Asian tiger mosquito Aedes albopictus (Skuse), is one of the most invasive mosquito species worldwide. In Mexico it is now recorded in 12 states and represents a serious public health problem, given the recent introduction of Chikungunya on the southern border. The aim of this study was to analyze the population genetics of A. albopictus from all major recorded foci, and model its ecological niche. Niche similarity with that from its autochthonous distribution in Asia and other invaded countries were analyzed and its potential future expansion and potential human exposure in climate change scenarios measured. We analyzed 125 sequences of a 317. bp fragment of the cyt b gene from seven A. albopictus populations across Mexico. The samples belong to 25 haplotypes with moderate population structuring (Fst. = 0.081, p

Vector-borne transmission of Trypanosoma cruzi (VBTTc) is dependent on the concomitant interaction between biological and environmental hazard over the entire landscape, and human vulnerability. Representations and practices of health-disease-care-seeking and territorial appropriation and use were analyzed for VBTTc in a qualitative ethnographic study in the Zoh-Laguna landscape, Campeche, Mexico. In-depth interviews and participatory observation explored representations and practices regarding ethno-ecological knowledge related to vector-transmission, health-disease-care-seeking, and land use processes. The population has a broad knowledge of biting insects, which they believe are all most abundant in the rainy season; the community´s proximity to natural areas is perceived as a barrier to control their abundance. Triatomines are mostly recognized by men, who have detailed knowledge regarding their occurrence and association with mammals in non-domestic fragments, where they report being bitten. Women emphasize the dermal consequences of triatomine bites, but have little knowledge about the disease. Triatomine bites and the chinchoma are "normalized" events which are treated using home remedies, if at all. The neglected condition of Chagas disease in Mexican public health policies, livelihoods which are dependent on primary production, and gender-related knowledge (or lack thereof) are structural circumstances which influence the environment and inhabitants´ living conditions; in turn, these trigger triatomine-human contact. The most important landscape practices producing vulnerability are the activities and mobility within and between landscape fragments causing greater exposure of inhabitants primarily in the dry season. A landscape approach to understanding vulnerability components of VBTTc from health-disease-care-seeking perspectives and based on territorial appropriation and use, is essential where there is continuous movement of vectors between and within all habitats. An understanding of the structural factors which motivate the population´s perceptions, beliefs, and practices and which create and maintain vulnerability is essential to develop culturally relevant and sustainable community-based VBTTc prevention and control.

Summary The wide geographical distribution of Triatoma dimidiata, one of the three major vectors of Chagas disease, ranges from Mexico to northern Peru. Since this species occupies a great diversity of artificial and natural ecotopes, its eradication is extremely difficult. In order to assist control efforts, we used chromosome analyses and DNA amount as taxonomic markers to study genetic variability in populations of T. dimidiata from Mexico, Guatemala, El Salvador and Colombia. We differentiated three groups or cytotypes defined by characteristic chromosome C-banding patterns and genome size measured by flow cytometry. The three cytotypes are restricted to different geographic locations. Cytotype 1 occurs in Mexico (excluding Yucatan), Guatemala (excluding Peten), El Salvador and Colombia. Cytotype 2 occurs in Yucatan and cytotype 3 occurs in Peten. Cytotype 1, commonly associated with domestic and peridomestic environments but also inhabiting sylvatic ecotopes, is the most widespread and with major epidemiological significance. In contrast, the Yucatan cytotype inhabits wild ecotopes but increasingly enters houses, while the Peten cytotype appears exclusively sylvatic. We suggest that these cytotypes represent cryptic species of T. dimidiata with different epidemiological relevance as Chagas disease vectors. Poor ability to colonize human dwellings, together with their restricted geographic distribution, indicate that the Yucatan and Peten putative species probably have much less epidemiological significance than cytotype 1. Thus, the genetic markers we describe are powerful tools to differentiate cryptic species in T. dimidiata with different epidemiological significance, contributing to planning the most effective control measures. La vaste distribution geographique de Triatoma dimidiate, un des trois principaux vecteurs de la maladie de Chagas, sevi de Mexico au nord du Perou. Parce que cette espece occupe une grande diversite d'habitat compose de terrain artificiel et d'ecotopes naturels, son eradication est extremement difficile. Dans le but de contribuer aux efforts de controle, nous avons utilise des analyses chromosomiques et les quantites d'ADN comme marqueurs taxonomiques pour etudier la variabilite genetique dans les populations de T. dimidiate de Mexico, Guatemala, Salvador et Colombie. Nous avons distingue trois groupes ou cytotypes definis par les profils caracteristiques en bandes C du chromosome et la taille du genome mesuree par flow cytometrie. Les trois cytotypes sont confines dans des locations geographiques differentes. Le cytotype 1 sevi a Mexico (a l'exception du Yucatan), au Guatemala (a l'exception de Peten), au Salvador et en Colombie. Le cytotype 2 sevi dans le Yucatan et le cytotype 3 a Peten. Le cytotype 1, communement associea l'environnement domestique et peri domestique mais aussi a des ecotopes sylvatiques habites, est le plus prevalant avec une epidemiologie majeure significative. Par contre, le cytotype du Yucatan sevi dans les ecotopes sauvages mais de plus en plus entre dans les maisons, alors que le cytotype de Peten semble etre essentiellement sylvatique. Nous suggerons donc que ces cytotypes representent des especes cryptiques de T. dimidiate avec differentes importances epidemiologiques en tant que vecteurs de la maladie de Chagas. Le peu d'habilitea coloniser les habitations humaines ainsi que leur distribution geographiques restreintes indiquent que les putatives especes du Yucatan et de Peten ont probablement peu de signification epidemiologique par rapport au cytotype 1. Les marqueurs genetiques que nous decrivons sont donc de puissants outils pour differencier des especes cryptiques de T. dimidiate, pouvant ainsi contribuer a planifier des mesures de controle plus efficaces. La amplia distribucion del Triatoma dimidiata, uno de los tres vectores principales de la enfermedad de Chagas, se extiende desde Mejico al norte de Peru. Puesto que esta especie ocupa una gran diversidad de ecotopos artificiales y naturales, su erradicacion es extremadamente dificil. Con el fin de ayudar en los esfuerzos de control, utilizamos el analisis cromosomico y la cantidad de ADN como marcadores taxonomicos para estudiar la variabilidad genetica en poblaciones de T. Dimidiata provenientes de Mejico, Guatemala, El Salvador y Colombia. Diferenciamos tres grupos o citotipos, definidos por bandeo cromosomico y patron de bandas C caracteristicos y por el tamano de su genoma, medido por citometria de flujo. Los tres citotipos estaban restringidos a diferentes areas geograficas. El citotipo 1 ocurria en Mejico (excluyendo Yucatan), Guatemala (excluyendo Peten), El Salvador y Colombia. El citotipo 2 ocurria en Yucatan y el citotipo 3 en Peten. El citotipo 1, comunmente asociado con ambientes domesticos y peridomesticos, pero que tambien habita ecotipos selvaticos, es el mas extendido y el que mayor significado epidemiologico tiene. En contraste, el citotipo de Yucatan habita ecotopos salvajes, pero cada vez con mayor frecuencia entra en las casas, mientras que el citotipo Peten es exclusivamente selvatico. Sugerimos que estos citotipos representan la especie criptica de T. Dimidiata con diferente relevancia epidemiologica como vectores de la enfermedad de Chagas. Su escasa habilidad para colonizar viviendas humanas, junto con su distribucion geografica restringida, indican que las especies putativas de Yucatan y Peten probablemente tienen un significado epidemiologico muchisimo menor que el citotipo 1. Por lo tanto, los marcadores geneticos que describimos son herramientas poderosas para diferenciar entre especies cripticas de T. dimidiata, contribuyendo a planear medidas de control mas efectivas.

Triatoma dimidiata is one of the major vectors of Chagas disease in Latin America. Its range includes Mexico, all countries of Central America, Colombia, and Ecuador. In light of recent genetic analysis suggesting that the possible origin of this species is the Yucatan peninsula, we have analyzed populations from the state of Yucatan, San Luis Potosi, and Veracruz in Mexico, and a population from the southern region of the Yucatan peninsula located in Northern Guatemala, the region of El Peten. Classical morphometry including principal component, discriminant, sexual dimorphism, and wing asymmetry was analyzed. San Luis Potosi and Veracruz populations were indistinguishable while clearly separate from Yucatan and Peten populations. Despite important genetic differences, Yucatan and Peten populations were highly similar. Yucatan specimens were the smallest in size, while females were larger than males in all populations. Only head characters were necessary to distinguish population level differences, although wing fluctuating asymmetry was present in all populations. These results are discussed in light of recent findings suggesting genetic polymorphism in most populations of Triatoma dimidiata south of Chiapas to Ecuador.

Chagas disease caused by infection with Trypanosoma cruzi Chagas (Kinetoplastida: Trypanosomatidae) is widespread in Mexico, transmitted by various triatomine bugs (Hemiptera: Reduviidae). The only domestic vector in Cuernavaca (population 650 000) is Triatoma pallidipennis (Stahl) with T. cruzi seroprevalence ranging from 1% to 9% in the resident human population. We surveyed possible risk factors for T. pallidipennis infestation at Cuernavaca (altitude 1200-2200 m) on south-western slopes of the Sierra Madre Occidental. This metropolitan area (with five administrative counties) has rapid urbanization, forested environs and proliferation of 'weekend housing' for visitors from Mexico City, 60 km to the north. To assess factors associated with T. pallidipennis infestation, we first stratified Cuernavaca by altitude and by socio-economic status of population catchment units (PCUs). Within each PCU, one to three blocks were chosen for cluster sampling (three houses/block) and information about Chagas disease was distributed. After obtaining signed consent from householders, representative houses were routinely and opportunistically inspected for T. pallidipennis and surveyed for demographic, economic, physical and other potential risk factors. Of the 1129 houses assessed, T. pallidipennis was found in 4.1% (range 3.0-6.8% per county) and the T. cruzi infection rate was approximately 50% in bugs. Rates of house infestation in poor PCUs were double those in higher socio-economic strata (odds ratio 2.12, confidence interval 1.03-4.3), with >4-fold greater crowding index of T. pallidipennis. The bug density index was inversely correlated with PCU altitude and socio-economic category (altitude of homes being associated with prosperity), while the bug colonization index (presence of nymphs indicating breeding) did not vary significantly across the PCU categories, but did vary according to altitude. Multivariate regression analysis showed that the most significant risk factors associated with T. pallidipennis infestation were lower altitude (linked with lower socio-economic status), garden area >80 m(2), dogs at liberty to enter the house, occurrence of squirrels and opossums around the house, presence of pigs in the surrounding area and having at least one of the adjacent lots empty (unconstructed). Householders who had received information about Chagas disease comprised 33% from infested houses (14/42) but only 15% from non-infested houses (148/984). Hence, the awareness of Chagas disease was significantly associated with having a bug-free house (P < 0.01). When shown specimens of T. pallidipennis, the proportions of householders who recognized them were 78% from infested houses but only 29% of those with uninfested houses. Given the low infestation rates and the high capacity of the population to act appropriately once they have received information regarding this disease and its vector, relevant health education is expected to have a significant impact on triatomine control in this metropolitan area.

In Mexico, Triatoma longipennis (Usinger), Triatoma picturata (Usinger), and Triatoma pallidipennis (Stal), primary Chagas disease vector species of the phyllosoma complex, were analyzed by randomly amplified polymorphic DNA (RAPD). Sixteen decametric primers resolved individual profiles not identical, but partially discriminative between species. Analysis based on pairwise presence/absence comparisons between the three species was performed using three primers and two outgroup species Triatoma infestans (Klug) and Triatoma barberi (Usinger). Fifty-three bands in total were scored, although only two bands were constant among the three phyllosoma complex species. Two other bands were constant only for T. longipennis and T. picturata together, and not present in T. pallidipennis. Neighbor Joining tree and the multiple correspondence analysis discriminated T. pallidipennis clearly from the other two species, although there was overlap between T. longipennis and T. picturata. The results indicate a close relationship between the studied species and support the hypothesis of their recent evolution. The suitability of RAPD to discern populations within the species is discussed.

Numerous recent studies have illuminated global distributions of human cases of dengue and other mosquito-transmitted diseases, yet the potential distributions of key vector species have not been incorporated integrally into those mapping efforts. Projections onto future conditions to illuminate potential distributional shifts in coming decades are similarly lacking, at least outside Europe. This study examined the global potential distributions of Aedes aegypti and Aedes albopictus in relation to climatic variation worldwide to develop ecological niche models that, in turn, allowed anticipation of possible changes in distributional patterns into the future. Results indicated complex global rearrangements of potential distributional areas, which—given the impressive dispersal abilities of these two species—are likely to translate into actual distributional shifts. This exercise also signalled a crucial priority: digitization and sharing of existing distributional data so that models of this sort can be developed more rigorously, as present availability of such data is fragmentary and woefully incomplete.

Ecologic niche modeling may improve our understanding of epidemiologically relevant vector and parasitereservoir distributions. We used this tool to identify host relationships of Triatoma species implicated in transmission of Chagas disease. Associations have been documented between the protracta complex (Triatoma: Triatominae: Reduviidae) with packrat species (Neotoma spp.), providing an excellent case study for the broader challenge of developing hypotheses of association. Species pairs that were identified coincided exactly with those in previous studies, suggesting that local interactions between Triatoma and Neotoma species and subspecies have implications at a geographic level. Nothing is known about sylvatic associates of T. barberi, which are considered the primary Chagas vector in Mexico; its geographic distribution coincided closely with that of N. mexicana, suggesting interaction. The presence of this species was confirmed in two regions where it had been predicted but not previously collected. This approach may help in identifying Chagas disease risk areas, planning vector-control strategies, and exploring parasite-reservoir associations for other emerging diseases. hagas disease is caused by the parasitic protozoan Trypanosoma cruzi and transmitted by blood-feeding insects in the family Reduviidae, subfamily Triatominae. Chagas disease is an important cause of illness and death throughout the Americas, affecting 16–18 million persons. While an estimated 100 million persons in 21 countries in the New World live in endemic areas and are at risk for infection, the disease is principally a zoonotic infection, in which sylvatic mammals serve as reservoir hosts and zoophilic triatomine species as vectors. The protracta species group consists of seven species (Triatoma protracta, T. peninsularis, T. sinaloensis, T. neotomae, T. barberi, T. nitida, and T. incrassata); T. protracta contains five subspecies: T. p. protracta, T. p. woodi, T. p. navajoensis, T. p. zacatecensis, and T. p. nahuatlae (1,2). This group is restricted to the southwestern United States and Mexico. Previous studies have demonstrated high host specificity in this species group, involving woodrats or packrats (Neotoma spp.) (1). Whereas host associations of Triatoma are often complex, the protracta group shows remarkable host specificity and geographic distributions suggestive of host-ectoparasite cospeciation. A new tool in the study of geographic phenomena in ecology and systematics is ecologic niche modeling of primary occurrence data (data placing a particular species in a particular site) (3). In general, the approach involves a machine-learning algorithm for discovering associations between pointoccurrence data and sets of electronic maps summarizing environmental/ecologic dimensions that may or may not be important in limiting species’ geographic distributions. These associations constitute an approximation of species’ fundamental ecologic niches (the conjunction of ecologic conditions in which a species is able to maintain populations without immigration) (4) and hence provide a basis for understanding numerous ecologic and geographic phenomena related to species distributions. We applied ecologic niche modeling to identify host relationships of Triatoma species and subspecies implicated in the transmission of Chagas disease. Previous studies by Ryckman (1) provide an ideal test case: hypotheses of association developed based on the modeling approach can be tested independently by using associations identified in Ryckman’s detailed field studies. If successful, this approach would be invaluable in identifying host relationships for species for which detailed information is not available, for stratifying Chagas disease risk areas, and for planning the operational aspects of vector control strategies. Methods

Immune modulation of Plasmodium vivax and P. falciparum gametocytes occurs over the course of erythrocytic infection. The response is linked to proliferative and inflammatory responses, which may be stimulated by stage-specific gametocyte proteins. Stage-specific exoantigens were purified from supernatants of P. falciparum and P. vivax gametocyte cultures, and either primary or secondary postinfection lymphocytes were stimulated for proliferation. Five of 25 exoantigens purified from P. falciparum gametocyte cultures and 6 of 28 exoantigens isolated from P. vivax were gametocyte stage specific. Metabolic labeling of soluble P. falciparum gametocyte proteins confirmed synthesis and secretion of 5 stage-specific exoantigens, with molecular masses of 118, 62, 52, 37, and 33 kDa. Purified gametocyte exoantigens within the range of 50 to 100 kDa stage-specifically stimulated proliferation of lymphocytes from postprimary P. falciparum infections, and from postprimary and secondary P. vivax infection patients with homologous purified exoantigens. T-cell receptor (TCR)gammadelta+, and CD3+ CD8+ and CD3+ CD4- CD8- T cells were specifically upregulated from P. falciparum primary- and P. vivax secondary-infection lymphocytes, respectively, using gametocyte stage-specific exoantigens. CD25+ was the major activation marker expressed by CD3+ and gammadelta T cells when stimulated with gametocyte exoantigens. None of the T cell markers was significantly upregulated using gametocyte stage-specific exoantigens with primary-infection P. vivax lymphocytes.

Randomly amplified polymorphic DNA (RAPD) has emerged as an effective genetic marker for analysis of Trypanosoma cruzi population variability. This method has been used to study the genetic variability of Mexican T. cruzi stocks and to relate these results to previous classifications. High clonal diversity was observed among the Mexican populations: 24 RAPD types were scored among 56 stocks analyzed. Only two stocks (3.6%) belonged to the T. cruzi II lineage, while all others belonged to T. cruzi I. The robustness of these clusters was statistically highly significant. Mexican T. cruzi I stocks formed a homogeneous group with reduced genetic distances among its members. Parasites from this group were isolated from both domestic and sylvatic cycles over a broad geographic area in Mexico. The two Mexican stocks classified as T. cruzi II (isolated from sylvatic cycles) were of the same RAPD type, although they were not closely related to the three reference T. cruzi II stocks circulating in domestic cycles in Argentina, Brazil, Bolivia, and Chile. These stocks were also unrelated to the formerly named Zymodeme III.

The niche conservatism hypothesis states that related species diverge in niche characteristics at lower rates than expected, given their lineage divergence. Here we analyze whether niche conservatism is a common pattern among vector species (Hemiptera: Reduviidae: Triatominae) of Trypanosoma cruzi that inhabit North and Central America, a highly heterogeneous landmass in terms of environmental gradients. Mitochondrial and nuclear loci were used in a multi-locus phylogenetic framework to reconstruct phylogenetic relationships among species and estimate time of divergence of selected clades to draw biogeographic inferences. Then, we estimated similarity between the ecological niche of sister species and tested the niche conservatism hypothesis using our best estimate of phylogeny. Triatoma is not monophyletic. A primary clade with all North and Central American (NCA) triatomine species from the genera Triatoma, Dipetalogaster, and Panstrongylus, was consistently recovered. Nearctic species within the NCA clade (T. p. protracta, T. r. rubida) diverged during the Pliocene, whereas the Neotropical species (T. phyllosoma, T. longipennis, T. dimidiata complex) are estimated to have diverged more recently, during the Pleistocene. The hypothesis of niche conservatism could not be rejected for any of six sister species pairs. Niche similarity between sister species best fits a retention model. While this framework is used here to infer niche evolution, it has a direct impact on spatial vector dynamics driven by human population movements, expansion of transportation networks and climate change scenarios., Author Summary Knowledge regarding the evolutionary history of insect vectors of pathogens is essential to design precise and appropriately integrated control strategies, since species' dispersal and invasive capacity are key components to prevent human-vector interaction. Given several well-known invasive or dispersal events of Triatominae, and their adaptive capacity to colonize modified habitats, the question arises whether niche conservatism (i.e. the limited capacity for niche differentiation within clades) across sister species could influence distribution patterns. Niche conservatism is herein analyzed across the highly heterogeneous landmass of North and Central America where most species complexes of the Triatominae are vectors of Trypanosoma cruzi. Since phylogenetic relationships among and within triatomine species complexes are still ill-defined, we first used a multi-locus phylogenetic analysis using mitochondrial and nuclear loci to validate relationships between species. Similarity of the ecological niche between six sister species' pairs was used to test the niche conservatism hypothesis. The results provide robust evidence of monophyly for North and Central America triatomine species. The hypothesis of niche conservatism was not rejected for any sister species pair, independently of niche width, distribution range, biogeographic affinity, or environmental heterogeneity; niche similarity correlated inversely with taxa divergence and according to a retention model.

Mexico has 18 species of Triatomine bugs (Hemiptera: Reduviidae) reported to be vectors of Trypanosoma cruzi. Chagas Disease is widespread in Mexico, with up to 3.5% seropositivity of human transfusion blood. The State of Oaxaca has the longest history of endemic Chagas Disease, based on acute and chronic case reports, and of entomological surveys in the country. However, the State health care services need more information on current risks of vector transmission. In order to identify and characterize areas of transmission in Oaxaca and to stratify the vector potential, the distribution of domestic Triatominae was surveyed during 1996-98 in collaboration with the primary health care services and local communities. Villages were studied in 11% of 570 municipalities in Oaxaca. Eight triatomine species were found in domestic and peri-domestic habitats: Triatoma barberi Usinger, T. bolivari Carcavallo et al., T. dimidiata (Latreille), T. mazzottii Usinger, T. nitida Usinger, T. pallidipennis (Stal), T. phyllosoma (Burmeister) and Rhodnius prolixus Stal. For each triatomine species in Oaxaca, the range of distribution and habitat characteristics are described. Habitat partitioning, principally based on altitude and mean annual precipitation, limited the overlap of distribution between species. Relatively consistent altitude of human settlements facilitates the dispersion of individual species within microregions. Entomological indices of house infestation were used to estimate that approximately 50% of the human population (1,874,320 inhabitants) would be at risk of vector transmission, with a minimum of 134,320 infected people and 40,280 chronic cases of Chagas Disease currently in Oaxaca.

Policy Platform A Regulatory Structure for Working with Genetically Modified Mosquitoes: Lessons from Mexico Janine M. Ramsey 1 , J. Guillermo Bond 1 , Maria Elena Macotela 1 , Luca Facchinelli 2,3 , Laura Valerio 2,4 , David M. Brown 5 , Thomas W. Scott 2 , Anthony A. James 5,6 * 1 Centro Regional de Investigacio´n en Salud Pu´blica, Instituto Nacional de Salud Pu´blica, Tapachula, Chiapas, Me´xico, 2 Department of Entomology, University of California, Davis, California, United States of America, 3 Department of Experimental Medicine, Functional Genomics Center, University of Perugia, Perugia, Italy, 4 Pasteur Institute–Cenci Bolognetti Foundation, University of Rome Sapienza, Rome, Italy, 5 Department of Microbiology and Molecular Genetics, University of California, Irvine, California, United States of America, 6 Department of Molecular Biology and Biochemistry, University of California, Irvine, California, United States of America Introduction Sustainable and effective control of dengue is hampered due to a number of factors, including the lack of evidence- based, locally relevant interventions; insuf- ficient information regarding key compo- nents of virus transmission and vector ecology; failure to implement precise and efficient surveillance systems; inefficient healthcare systems; ineffective health pro- motion and outreach resulting in lack of community dialogue and participation; and a paucity of efficient diagnostic strategies and clinical attention [1]. In- creased research efforts in response to the complexity of this problem have focused on the development of novel technologies that would enhance existing tools for vector-borne disease prevention [2–4]. Genetic strategies to reduce or replace mosquito populations and thereby inter- rupt transmission of dengue viruses are among the new approaches being consid- ered [5–7]. Many of these approaches take advantage of molecular genetic tools to engineer traits that cause lethal pheno- types or confer resistance to the pathogen in the mosquito. Genetic strategies are being advanced through a series of overlapping domains that inform the decision making on feasibility, safety, efficacy, and acceptabil- ity. Although the need to focus on science- based regulation using a risk-assessment framework is gaining support [8], there has been a relative lack of attention on broader community regulations that are explicitly or indirectly required to bring a genetics-based product to the field [3,9– 11]. An evidence-based approach would facilitate the integration, efficacy, and acceptability of policy for an intervention strategy. We addressed the regulatory challenges associated with testing a strain of Aedes aegypti engineered to result in population suppression in contained field trials in southwestern Mexico [12]. This large research effort (designated hereafter as the ‘‘Project’’) combined elements of scientific and social discovery and devel- opment as the basis for moving a new technology from the laboratory to the field. Unlike the rollout of other public- health products such as drugs, vaccines, and insecticides, no pipelines exist to move candidate genetically modified mosquitoes (GMMs) from the laboratory through safety and efficacy trials to field deploy- ment. This lack of a preexisting structure made it necessary for the scientists in the Project to play critical, unbiased roles in formulating the product development pathway. The challenge offered a unique opportunity for potential end users and beneficiaries of the technology to be involved from the beginning in product discovery and development. This ap- proach ensures that requirements for safety and efficacy are included as design features engineered into the modified mosquito strains [13]. It is incumbent on the researchers to identify gaps and assist in development of regulatory norms that should be applied to the products they create. These norms include not only statutory regulations but also a broader regulatory environment that addresses the needs and concerns of all communities in which the product will be applied. We describe here the regulatory and social structures used for obtaining approvals in Mexico. This review of our approach is intended to stimulate analysis and dia- logue that will help refine regulatory practices of genetic-based strategies for vector-borne disease control. Regulatory Domains for the Discovery and Development of Genetically Modified Mosquitoes An initial challenge of the Project was identifying relevant communities [14]. No consensus existed as to what comprises a relevant community to engage for a GMMs research project, nor were there any widely accepted methods for identify- ing their members. We adopted a defini- tion (modified from [15]) in which the community consists of all those individuals who share the identified risks and/or will benefit from the outcome of the proposed research project. In this context, the community coalesces as a result of the project and evolves continuously as it progresses through conceptualization, dis- cussion, and implementation [16]. The community is formed ultimately by those individuals, groups, organizations, and agencies that have legitimate interest in the research, and therefore they must be engaged in an effective and timely man- ner. Successfully moving a novel technology from the laboratory to practical application Citation: Ramsey JM, Bond JG, Macotela ME, Facchinelli L, Valerio L, et al. (2014) A Regulatory Structure for Working with Genetically Modified Mosquitoes: Lessons from Mexico. PLoS Negl Trop Dis 8(3): e2623. doi:10.1371/journal.pntd.0002623 Editor: Jesus G. Valenzuela, National Institute of Allergy and Infectious Diseases, United States of America Published March 13, 2014 Copyright: s 2014 Ramsey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research and Policy for Infectious Disease Dynamics program of the Science and Technology Directory, Department of Homeland Security; Fogarty International Center, National Institutes of Health; Pasteur Institute – Cenci Bolognetti Foundation; Foundation for the National Institutes of Health through the Grand Challenges in Global Health (GCGH) initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare no conflict of interest. * E-mail: aajames@uci.edu PLOS Neglected Tropical Diseases | www.plosntds.org March 2014 | Volume 8 | Issue 3 | e2623

Ecological niche models are useful tools to infer potential spatial and temporal distributions in vector species and to measure epidemiological risk for infectious diseases such as the Leishmaniases. The ecological niche of 28 North and Central American sand fly species, including those with epidemiological relevance, can be used to analyze the vector's ecology and its association with transmission risk, and plan integrated regional vector surveillance and control programs. In this study, we model the environmental requirements of the principal North and Central American phlebotomine species and analyze three niche characteristics over future climate change scenarios: i) potential change in niche breadth, ii) direction and magnitude of niche centroid shifts, iii) shifts in elevation range. Niche identity between confirmed or incriminated Leishmania vector sand flies in Mexico, and human cases were analyzed. Niche models were constructed using sand fly occurrence datapoints from Canada, USA, Mexico, Guatemala and Belize. Nine non-correlated bioclimatic and four topographic data layers were used as niche components using GARP in OpenModeller. Both B2 and A2 climate change scenarios were used with two general circulation models for each scenario (CSIRO and HadCM3), for 2020, 2050 and 2080. There was an increase in niche breadth to 2080 in both scenarios for all species with the exception of Lutzomyia vexator. The principal direction of niche centroid displacement was to the northwest (64%), while the elevation range decreased greatest for tropical, and least for broad-range species. Lutzomyia cruciata is the only epidemiologically important species with high niche identity with that of Leishmania spp. in Mexico. Continued landscape modification in future climate change will provide an increased opportunity for the geographic expansion of NCA sand flys' ENM and human exposure to vectors of Leishmaniases., Author Summary The present study models the niche of the most abundant sand fly species in North and Central America, including all proven and incriminated vectors of Leishmaniases, an important neglected tropical disease of the region. The expansion and elevation or centroid shifts of the species' niche are modeled for extreme (A2) and conservative (B2) climate change scenarios to 2020, 2050 and 2080. In climate change scenarios, models predict significant niche breadth changes in geographic space, principally in temperate sand fly species, while elevation shifts occur principally in tropical, and greatest, in vector species. Niche centroid shifts for individual species were predominately to the northwest, and secondarily to the northeast. The highest proportion of human population at-risk for contact with a vector species was with Lutzomyia diabolica and Lutzomyia shannoni. Despite the fact that Lutzomyia olmeca olmeca is the only confirmed vector species in Mexico, the present study demonstrates a significant niche identity between Leishmania spp. and Lutzomyia cruciata.

Transgenic strains of Aedes aegypti have been engineered to help control transmission of dengue virus. Although resources have been invested in developing the strains, we lack data on the ecology of mosquitoes that could impact the success of this approach. Although studies of intra-specific competition have been conducted using Ae. aegypti larvae, none of these studies examine mixed age cohorts at densities that occur in the field, with natural nutrient levels. Experiments were conducted in Mexico to determine the impact of direct and delayed density dependence on Ae. aegypti populations. Natural water, food, and larval densities were used to estimate the impacts of density dependence on larval survival, development, and adult body size. Direct and delayed density-dependent factors had a significant impact on larval survival, larval development, and adult body size. These results indicate that control methods attempting to reduce mosquito populations may be counteracted by density-dependent population regulation.

We demonstrate the utility of models as aids in the design and development of experiments aimed at measuring the effects of proposed vector population control strategies. We describe the exploration of a stochastic, age-structured model that simulates field cage experiments that test the ability of a female-killing strain of the mosquito Aedes aegypti (L.) to suppress a wild-type population. Model output predicts that choices of release ratio and population size can impact mean extinction time and variability in extinction time among experiments. We find that unless fitness costs are >80% they will not be detectable in experiments with high release ratios. At lower release ratios, the predicted length of the experiment increases significantly for fitness costs >20%. Experiments with small populations may more accurately reflect field conditions, but extinction can occur even in the absence of a functional female-killing construct because of stochastic effects. We illustrate how the model can be used to explore experimental designs that aim to study the impact of density dependence and immigration; predictions indicate that cage population eradication may not always be obtainable in an operationally realistic time frame. We propose a method to predict the extinction time of a cage population based on the rate of population reduction with the goal of shortening the duration of the experiment. We discuss the model as a tool for exploring and assessing the utility of a wider range of scenarios than would be feasible to test experimentally because of financial and temporal restraints.

The low dispersal capacity of sand flies could lead to population isolation due to geographic barriers, climate variation, or to population fragmentation associated with specific local habitats due to landscape modification. The phlebotomine sand fly Lutzomyia cruciata has a wide distribution throughout Mexico and is a vector of Leishmania mexicana in the southeast. The aim of this study was to evaluate the genetic diversity, structure, and divergence within and among populations of Lu. cruciata in the state of Chiapas, and to infer the intra-specific phylogeny using the 3' end of the mitochondrial cytochrome b gene. We analyzed 62 sequences from four Lu. cruciata populations and found 26 haplotypes, high genetic differentiation and restricted gene flow among populations (Fst=0.416, Nm=0.701, p

Background A genetically-engineered strain of the dengue mosquito vector Aedes aegypti, designated OX3604C, was evaluated in large outdoor cage trials for its potential to improve dengue prevention efforts by inducing population suppression. OX3604C is engineered with a repressible genetic construct that causes a female-specific flightless phenotype. Wild-type females that mate with homozygous OX3604C males will not produce reproductive female offspring. Weekly introductions of OX3604C males eliminated all three targeted Ae. aegypti populations after 10–20 weeks in a previous laboratory cage experiment. As part of the phased, progressive evaluation of this technology, we carried out an assessment in large outdoor field enclosures in dengue endemic southern Mexico. Methodology/Principal Findings OX3604C males were introduced weekly into field cages containing stable target populations, initially at 10∶1 ratios. Statistically significant target population decreases were detected in 4 of 5 treatment cages after 17 weeks, but none of the treatment populations were eliminated. Mating competitiveness experiments, carried out to explore the discrepancy between lab and field cage results revealed a maximum mating disadvantage of up 59.1% for OX3604C males, which accounted for a significant part of the 97% fitness cost predicted by a mathematical model to be necessary to produce the field cage results. Conclusions/Significance Our results indicate that OX3604C may not be effective in large-scale releases. A strain with the same transgene that is not encumbered by a large mating disadvantage, however, could have improved prospects for dengue prevention. Insights from large outdoor cage experiments may provide an important part of the progressive, stepwise evaluation of genetically-engineered mosquitoes., Author Summary The absence of a commercially-available dengue vaccine or anti-viral drug makes control of Aedes aegypti, the principal dengue mosquito vector, the only available method to prevent this disease. Sustained, effective application of vector control, however, has been difficult and this led to the call for innovative strategies, including genetic approaches. Here, the authors investigated the ability of a genetically-engineered strain of Ae. aegypti to eliminate wild mosquito populations in large outdoor cages. Females of the engineered strain cannot fly and, therefore, cannot mate or take blood meals necessary to lay viable eggs. Wild females that mate with genetically-engineered males, therefore, will not produce reproductive female offspring. In this study, although population reductions were detected in 4 of 5 field cages, none of the wild mosquito populations were eliminated. A mating disadvantage for genetically-engineered males appeared to account for a significant part of their fitness disadvantage. Results suggest that this specific strain may not be effective in a large-scale release and that new strains with the same or similar transgene, but improved mating performance, may be more effective for preventing dengue. Results also indicate that large outdoor cage experiments may provide valuable insights into the progressive, stepwise assessment of genetically-engineered mosquitoes.

Most Aedes aegypti dispersal studies have focused on females because of their central role in dengue virus transmission. Only a few mark-release-recapture (MRR) studies provided insights into male Ae. aegypti dispersal. To fill this knowledge gap, we conducted five male Ae. aegypti MRR experiments in a coastal village in southern Mexico. Small and large male cohorts were marked with fluorescent dusts, released outside buildings, and recaptures were carried out by using backpack aspirators. Recapture rates ranged between 0.35% and 6.55% and median distance traveled was 12-166 meters. A statistically significant difference in median distance traveled with large males dispersing farther than small ones was detected only in one experiment (MRR5: U = 3.5, P < 0.01). Male dispersal data will be useful for constructing and estimating parameter values and validating models that will be used to plan the most effective release strategies for genetically modified male Ae. aegypti.

Female Aedes aegypti are assumed to be primarily monandrous (i.e., mate only once in their lifetime), but true estimates of mating frequency have not been determined outside the laboratory. To assess polyandry in Ae. aegypti with first-generation progeny from wild mosquitoes, stable isotope semen-labeled males ((15)N or (13)C) were allowed to mate with unlabeled females in semifield enclosures (22.5 m(3)) in a dengue-endemic area in southern Mexico. On average, 14% of females were positive for both labels, indicating that they received semen from more than one male. Our results provide evidence of a small but potentially significant rate of multiple mating within a 48-hour period and provide an approach for future open-field studies of polyandry in this species. Polyandry has implications for understanding mosquito ecology, evolution, and reproductive behavior as well as genetic strategies for mosquito control.

Background Aedes aegypti, the ‘yellow fever mosquito’, is the primary vector to humans of dengue and yellow fever flaviviruses (DENV, YFV), and is a known vector of the chikungunya alphavirus (CV). Because vaccines are not yet available for DENV or CV or are inadequately distributed in developing countries (YFV), management of Ae. aegypti remains the primary option to prevent and control outbreaks of the diseases caused by these arboviruses. Permethrin is one of the most widely used active ingredients in insecticides for suppression of adult Ae. aegypti. In 2007, we documented a replacement mutation in codon 1,016 of the voltage-gated sodium channel gene (para) of Ae. aegypti that encodes an isoleucine rather than a valine and confers resistance to permethrin. Ile1,016 segregates as a recessive allele conferring knockdown resistance to homozygous mosquitoes at 5–10 µg of permethrin in bottle bioassays. Methods and Findings A total of 81 field collections containing 3,951 Ae. aegypti were made throughout México from 1996 to 2009. These mosquitoes were analyzed for the frequency of the Ile1,016 mutation using a melting-curve PCR assay. Dramatic increases in frequencies of Ile1,016 were recorded from the late 1990's to 2006–2009 in several states including Nuevo León in the north, Veracruz on the central Atlantic coast, and Yucatán, Quintana Roo and Chiapas in the south. From 1996 to 2000, the overall frequency of Ile1,016 was 0.04% (95% confidence interval (CI95) = 0.12%; n = 1,359 mosquitoes examined). The earliest detection of Ile1,016 was in Nuevo Laredo on the U.S. border in 1997. By 2003–2004 the overall frequency of Ile1,016 had increased ∼100-fold to 2.7% (±0.80% CI95; n = 808). When checked again in 2006, the frequency had increased slightly to 3.9% (±1.15% CI95; n = 473). This was followed in 2007–2009 by a sudden jump in Ile1,016 frequency to 33.2% (±1.99% CI95; n = 1,074 mosquitoes). There was spatial heterogeneity in Ile1,016 frequencies among 2007–2008 collections, which ranged from 45.7% (±2.00% CI95) in the state of Veracruz to 51.2% (±4.36% CI95) in the Yucatán peninsula and 14.5% (±2.23% CI95) in and around Tapachula in the state of Chiapas. Spatial heterogeneity was also evident at smaller geographic scales. For example within the city of Chetumal, Quintana Roo, Ile1,016 frequencies varied from 38.3%–88.3%. A linear regression analysis based on seven collections from 2007 revealed that the frequency of Ile1,016 homozygotes accurately predicted knockdown rate for mosquitoes exposed to permethrin in a bioassay (R2 = 0.98). Conclusions We have recorded a dramatic increase in the frequency of the Ile1,016 mutation in the voltage-gated sodium channel gene of Ae. aegypti in México from 1996 to 2009. This may be related to heavy use of permethrin-based insecticides in mosquito control programs. Spatial heterogeneity in Ile1,016 frequencies in 2007 and 2008 collections may reflect differences in selection pressure or in the initial frequency of Ile1,016. The rapid recent increase in Ile1,016 is predicted by a simple model of positive directional selection on a recessive allele. Unfortunately this model also predicts rapid fixation of Ile1,016 unless there is negative fitness associated with Ile1,016 in the absence of permethrin. If so, then spatial refugia of susceptible Ae. aegypti or rotational schedules of different classes of adulticides could be established to slow or prevent fixation of Ile1,016., Author Summary Pyrethroid insecticides prolong the opening of voltage-dependent sodium channels in insect nerves to produce instant paralysis and “knock-down.” Many insects have evolved knock-down resistance through nonsynonymous mutations that reduce pyrethroid binding in the channels. In 2006 we discovered one such mutation in the arbovirus mosquito vector Aedes aegypti, called Ile1,016, that confers very high knockdown resistance to the pyrethroid insecticide permethrin in mosquitoes homozygous for this mutation. We examined collections of Ae. aegypti from México during 1996–2009 and found that the overall Ile1,016 frequency increased from

Chagas disease represents one of the more significant public health problems in the Americas. Information regarding the genome and proteome of vectors and parasite, as well as their interactions, will be essential to develop specific and effective diagnostic and preventive tools. Advances that have contributed to the design, implementation, and efficacy of disease surveillance and control activities are reviewed. Genomic and proteomic information has contributed to a better understanding of vector distributions and dispersion, diversity, population dynamics, and control targets (populations and species). In addition, genomic and proteomic studies have impacted parasite diagnostics, Trypanosoma cruzi population dynamics, pharmacological treatment and knowledge of parasite-host interactions. Discussion of these contributions includes expectations for future basic and applied research questions.

The first U.S. ELISA test for T. cruzi antibodies was licensed by the Food and Drug Administration (FDA) on December 13, 2006. Blood banks have begun screening in absence of FDA recommendations for best implementation methods. We surveyed 2,029 blood donors at five California sites with three risk-based Chagas risk-screening questions. Semi-Markov models compared the cost-effectiveness of three testing strategies. 30% of donors screened positively. Screening all dominated doing nothing, being less costly, and saving more lives. The choice to "screen and test" compared with "testing all" varied by Chagas prevalence, "screening and testing" being cost-effective for high (0.004) and low (0.00004) prevalences, and "testing all" cost-effective for moderate risk (0.0004). It is cost-effective to screen by ELISA rather than do nothing. The best strategy depends on site-specific risk. Census estimates of Hispanics do not predict donor risk well. We suggest using our screening questions to determine risk level and most cost-effective testing strategy.

Background Among Chagas disease triatomine vectors, the largest genus, Triatoma, includes species of high public health interest. Triatoma dimidiata, the main vector throughout Central America and up to Ecuador, presents extensive phenotypic, genotypic, and behavioral diversity in sylvatic, peridomestic and domestic habitats, and non-domiciliated populations acting as reinfestation sources. DNA sequence analyses, phylogenetic reconstruction methods, and genetic variation approaches are combined to investigate the haplotype profiling, genetic polymorphism, phylogeography, and evolutionary trends of T. dimidiata and its closest relatives within Triatoma. This is the largest interpopulational analysis performed on a triatomine species so far. Methodology and Findings Triatomines from Mexico, Guatemala, Honduras, Nicaragua, Panama, Cuba, Colombia, Ecuador, and Brazil were used. Triatoma dimidiata populations follow different evolutionary divergences in which geographical isolation appears to have had an important influence. A southern Mexican–northern Guatemalan ancestral form gave rise to two main clades. One clade remained confined to the Yucatan peninsula and northern parts of Chiapas State, Guatemala, and Honduras, with extant descendants deserving specific status. Within the second clade, extant subspecies diversity was shaped by adaptive radiation derived from Guatemalan ancestral populations. Central American populations correspond to subspecies T. d. dimidiata. A southern spread into Panama and Colombia gave the T. d. capitata forms, and a northwestern spread rising from Guatemala into Mexico gave the T. d. maculipennis forms. Triatoma hegneri appears as a subspecific insular form. Conclusions The comparison with very numerous Triatoma species allows us to reach highly supported conclusions not only about T. dimidiata, but also on different, important Triatoma species groupings and their evolution. The very large intraspecific genetic variability found in T. dimidiata sensu lato has never been detected in a triatomine species before. The distinction between the five different taxa furnishes a new frame for future analyses of the different vector transmission capacities and epidemiological characteristics of Chagas disease. Results indicate that T. dimidiata will offer problems for control, although dwelling insecticide spraying might be successful against introduced populations in Ecuador., Author Summary Chagas disease is a serious parasitic disease of Latin America. Human contamination in poor rural or periurban areas is mainly attributed to haematophagous triatomine insects. Triatoma includes important vector species, as T. dimidiata in Central and Meso-America. DNA sequences, phylogenetic methods and genetic variation analyses are combined in a large interpopulational approach to investigate T. dimidiata and its closest relatives within Triatoma. The phylogeography of Triatoma indicates two colonization lineages northward and southward of the Panama isthmus during ancient periods, with T. dimidiata presenting a large genetic variability related to evolutionary divergences from a Mexican-Guatemalan origin. One clade remained confined to Yucatan, Chiapas, Guatemala and Honduras, with extant descendants deserving species status: T. sp. aff. dimidiata. The second clade gave rise to four subspecies: T. d. dimidiata in Guatemala and Mexico (Chiapas) up to Honduras, Nicaragua, Providencia island, and introduced into Ecuador; T. d. capitata in Panama and Colombia; T. d. maculipennis in Mexico and Guatemala; and T. d. hegneri in Cozumel island. This taxa distinction may facilitate the understanding of the diversity of vectors formerly included under T. dimidiata, their different transmission capacities and the disease epidemiology. Triatoma dimidiata will offer more problems for control than T. infestans in Uruguay, Chile and Brazil, although populations in Ecuador are appropriate targets for insecticide-spraying.

Because information about genome size in triatomines is scarce and contradictory, we performed DNA quantification by flow cytometry in 13 species belonging to five genera (Dipetalogaster, Eratyrus, Panstrongylus, Rhodnius, and Triatoma) to infer overall tendencies and phylogenetic associations. The results show that the haploid DNA content of the subfamily Triatominae varies nearly 4-fold, from < 0.7 pg in Rhodnius species (0.6 × 109 bp) to 2.7 pg in Triatoma delpontei (2.6 × 109 bp). Considering that triatomines present similar chromosome numbers, we suggest that genome size differences are the result of variation in the quantity of repetitive DNA sequences localized in hetero and euchromatin. Changes in heterochromatin are particularly important when considering populations or closely related species; in more distant taxa, euchromatic changes also play a role. Our analyses indicate that flow cytometry is a useful tool for population, taxonomic, and evolutionary studies in this subfamily. COL0007865

Plasmodium gametocytes are responsible for transmission from the vertebrate host to the mosquito. Plasmodium gametocytes undergo a complex cycle from asexual stages, through a poorly understood process characterized by expression of stage-specific proteins and adhesion molecules. Gametocytes are capable of inducing specific humoral IgG, and cellular responses, which include induction of TNFalpha, IFNgamma and gammadelta+ lymphocyte proliferation, in addition to immune responses to other stages of the parasite (sporozoite, exo-erythrocytic stages, erythrocytic stages). Although transmission-blocking vaccines against Plasmodium do not currently include components against the gametocytes (rather they focus on gametes, zygotes or ookinetes, stages which occur in the mosquito), further understanding of the mechanisms underlying gametocytogenesis and immune responses against these stages may provide additional strategies for more effective transmission inhibition.

A single village control trial for Triatoma pallidipennis and T. barberi was conducted using three synthetic pyrethroids (bifenthrin, cyfluthrin, and deltamethrin), evaluated as residual treatments in separate sectors, with complete coverage indoors and in peridomiciliary areas. Spray intervention was preceded by a preintervention entomological evaluation and household survey, followed by four postintervention evaluations at 1, 3, 6, and 12 mo of96% of houses. Overall preintervention adjusted infestation index was 38%, 17% of which represented intradomicile infestation. Dose verification using high-performance liquid chromatography (HPLC) demonstrated correct spray doses for all but deltamethrin treatments. There was between a 6- and 13-fold decrease in intradomicile live bug infestation for cyfluthrin- and bifenthrin-treated areas, resulting at 1 mo in 0 and 0.6% infestation, respectively. Intradomicile infestation recovered somewhat, terminating at 20 and 50% of preintervention levels at 12 mo, respectively, while peridomicile infestation recovered preintervention levels within 3-6 mo. Households with persistent live peridomiciliary infestation had 1.9 times the risk of having a persistent intradomiciliary infestation, while 80% of peridomicile infestations for both triatomine species were in houses not having a previous infestation. New or reinfestation of households did not occur consistent with a sylvan source, and unconstructed lots were not a significant source of bugs. Houses with persistent peridomicile infestation did represent a significant risk for surrounding uninfested houses by cluster analysis (P0.05). Along with the increased prevalence of T. cruzi infection after intervention, the data indicate that a sylvan reservoir source, probably peridomicile small rodent nests, represent the major risk factor for persistent and new infestations.

After three dogs died from acute Chagas cardiomyopathy at one location, an investigation was conducted of the home, garage, and grounds of the owner. A serologic study was conducted on stray dogs, and an ecologic niche model was developed to predict areas where the vector Tryiatoma gerstaeckeri might be expected.

The nucleotide sequences of the rDNA second internal transcribed spacer (ITS-2) of 31 populations of 12 and 3 species of the two main Triatominae tribes Triatomini and Rhodniini, including the most important Chagas disease vectors, were obtained. Sequence comparisons and parsimony, distance, and maximum-likelihood analyses indicate that ITS-2 is a useful marker for resolving supraspecific, specific, subspecific, and even sometimes population-level relationships in Triatominae. Results were markedly different between species of Triatomini and Rhodniini, suggesting polyphyly. Phylogenetic trees support an old divergence between South American and North-Central American Triatomini and query the validity of some genera (Dipetalogaster, Psammolestes). The very low sequence variation between species of the phyllosoma complex suggests that subspecific ranking would be more appropriate. Triatoma dimidiata proves to be a clearly differentiated species, with several populations evidencing a clinal variation along a north-south axis and a population from Yucatan showing differences consistent with specific status.

Triatoma barberi, T. dimidiata, T. longipennis, T. pallidipennis and T. picturata, all key Chagas disease vectors in Mexico, were analysed by multilocus enzyme electrophoresis (MLEE) at 17 putative loci. The majority of insect specimens studied were collected from domestic and peridomestic structures from multiple geographic locations while others were collected from sylvatic areas. T. barberi was the least polymorphic species ( P( 0.95) = 0.18), with polymorphism rates of the other species ranging from 0.29 to 0.50. T. barberi, a member of the protracta complex, clustered apart from the other studied species by Nei’s genetic distance with >1.36, and at least eight loci were found to be diagnostic for this species. T. dimidiata was more related to T. longipennis, T. pallidipennis and T. picturata (phyllosoma complex) than to T. barberi, with a genetic distance averaging 0.36 with the phyllosoma complex species. In contrast, the genetic distances between the three phyllosoma complex species were not significantly different from zero, and there were no species-specific loci differentiating among them. The results strongly support the grouping of these three species in one complex, separate from the two other species studied. © 2001 Elsevier Science B.V. All rights reserved.

The evolutionary history and times of divergence of triatomine bug lineages are estimated from molecular clocks inferred from nucleotide sequences of the small subunit SSU (18S) and the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA of these reduviids. The 18S rDNA molecular clock rate in Triatominae, and Prosorrhynchan Hemiptera in general, appears to be of 1.8% per 100 million years (my). The ITS-2 molecular clock rate in Triatominae is estimated to be around 0.4-1% per 1 my, indicating that ITS-2 evolves 23-55 times faster than 18S rDNA. Inferred chronological data about the evolution of Triatominae fit well with current hypotheses on their evolutionary histories, but suggest reconsideration of the current taxonomy of North America species complexes. (Résumé d'auteur)

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

Background: Triatoma dimidiata (Latreille) is a key vector complex of Trypanosoma cruzi, etiologic agent of Chagas disease, as it spans North, Central, and South America. Although morphological and genetic studies clearly indicate existence of at least five clades within the species, there has been no robust or systematic revision, or appropriate nomenclature change for species within the complex. Three of the clades (haplogroups) are distributed in Mexico, and recent evidence attests to dispersal of clades across previously "presumed"monotypic geographic regions. Evidence of niche conservatism among sister species of this complex suggests that geographic dispersal is possible for non-sympatric populations, although no information is available on the behavioural aspects of potential interclade interactions, for instance whether differentiation of chemical signaling or response to these signals could impede communication among the haplogroups. Methods: Volatiles emitted by disturbed bugs, Brindley's (BGs), and metasternal (MGs) glands were identified using solid-phase micro-extraction (SPME) and gas chromatography coupled mass spectrometry (GC-MS). Volatile compounds emitted by BGs and MGs, and those secreted by disturbed nymphs and adults, of the three Mexican T. dimidiata haplogroups were tested for avoidance behaviour by conspecific nymphs and adults using an olfactometer. Results: Triatoma dimidiata haplogroups all have three age-related alarm responses: absence of response by early stage nymphs, stage-specific response by 4-5th stage nymphs, and a shared 4-5th nymph and adult response to adult compounds. Disturbed bugs released 15 to 24 compounds depending on the haplogroup, among which were three pyrazines, the first report of these organoleptics in Triatominae. Isobutyric acid from BGs was the most abundant molecule in the response in all haplogroups, in addition to 15 (h1) to 21 (h2 and h3) MG compounds. Avoidance behaviour of disturbed bugs and volatiles emitted by BGs were haplogroup specific, while those from the MG were not. Conclusions: Discriminant and cluster analysis of BG +MG compounds indicate significant separation among the three haplogroups, while alarm response compounds were similar between h2 and h3, both distinct from h1. This latter haplogroup is ancestral phylogenetically to the other two. Our results suggest that alarm responses are a conserved behaviour in the Triatoma dimidiata complex. Fil: May Concha, Irving Jesus. Provincia de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Universidad Autónoma de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción; Argentina. Instituto Nacional de Salud Pública; México Fil: Rojas, Julio C.. El Colegio de la Frontera Sur; México Fil: Cruz López, Leopoldo. El Colegio de la Frontera Sur; México Fil: Ibarra-Cerdeña, Carlos N.. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzados; México Fil: Ramsey, Janine. Instituto Nacional de Salud Pública; México