Trypanosoma cruzi in Mexican Neotropical vectors and mammals: wildlife, livestock, pets, and human population.

OBJECTIVE: To provide primary evidence of Trypanosoma cruzi landscape genetics in the Mexican Neotropics. MATERIALS AND METHODS: Trypanosoma cruzi and discrete typing units (DTU) prevalence were analyzed in landscape communities of vectors, wildlife, livestock, pets, and sympatric human populations using endpoint PCR and sequencing of all relevant amplicons from mitochondrial (kDNA) and nuclear (ME, 18S, 24Sα) gene markers. RESULTS: Although 98% of the infected sample-set (N=2 963) contained single or mixed infections of DTUI (TcI, 96.2%) and TcVI (22.6%), TcIV and TcII were also identified. Sensitivity of individual markers varied and was dependent on host taxon; kDNA, ME and 18S combined identified 95% of infections. ME genotyped 90% of vector infections, but 60% of mammals (36% wildlife), while neither 18S nor 24Sα typed more than 20% of mammal infections. CONCLUSION: Available gene fragments to identify or genotype T. cruzi are not universally sensitive for all landscape parasite populations, highlighting important T. cruzi heteroge- neity among mammal reservoir taxa and triatomine species.

Species delimitation and integrative taxonomy of the Reithrodontomys mexicanus (Rodentia: Cricetidae) cryptic complex.

Species boundaries are difficult to establish in groups with very similar morphology. As an alternative, it has been suggested to integrate multiple sources of data to clarify taxonomic problems in taxa where cryptic speciation processes have been reported. This is the case of the harvest mouse Reithrodontomys mexicanus, which has a problematic taxonomy history as it is considered a complex species. Here, we evaluate the cryptic diversity of R. mexicanus using an integrative taxonomy approach in order to detect candidate lineages at the species level. The molecular analysis used one mitochondrial (cytb) and two nuclear (Fgb-I7 and IRBP) genes. Species hypotheses were suggested based on three molecular delimitation methods (mPTP, bGMYC, and STACEY) and cytb genetic distance values. Skull and environmental space differences between the delimited species were also tested to complement the discrimination of candidate species. Based on the consensus across the delimitation methods and genetic distance values, four species were proposed, which were mostly supported by morphometric and ecological data: R. mexicanus clade I, R. mexicanus clade IIA, R. mexicanus clade IIIA, and R. mexicanus clade IIIB. In addition, the evolutionary relationships between the species that comprise the R. mexicanus group were discussed from a phylogenetic approach. Our findings present important taxonomic implications for Reithrodontomys, as the number of known species for this genus increases. Furthermore, we highlight the importance of the use of multiple sources of data in systematic studies to establish robust delimitations between species considered taxonomically complex.

Geometric morphometrics and ecological niche modelling for delimitation of Triatoma pallidipennis (Hemiptera: Reduviidae: Triatominae) haplogroups.

A recent phylogenetic analysis of Triatoma pallidipennis, an important Chagas disease vector in Mexico, based on molecular markers, revealed five monophyletic haplogroups with validity as cryptic species. Here, we compare T. pallidipennis haplogroups using head and pronotum features, environmental characteristics of their habitats, and ecological niche modeling. To analyze variation in shape, images of the head and pronotum of the specimens were obtained and analyzed using methods based on landmarks and semi-landmarks. Ecological niche models were obtained from occurrence data, as well as a set of bioclimatic variables that characterized the environmental niche of each analyzed haplogroup. Deformation grids for head showed a slight displacement towards posterior region of pre-ocular landmarks. Greatest change in head shape was observed with strong displacement towards anterior region of antenniferous tubercle. Procrustes ANOVA and pairwise comparisons showed differences in mean head shape in almost all haplogroups. However, pairwise comparisons of mean pronotum shape only showed differences among three haplogroups. Correct classification of all haplogroups was not possible using discriminant analysis. Important differences were found among the environmental niches of the analyzed haplogroups. Ecological niche models of each haplogroup did not predict the climatic suitability areas of the other haplogroups, revealing differences in environmental conditions. Significant differences were found between at least two haplogroups, demonstrating distinct environmental preferences among them. Our results show how the analysis of morphometric variation and the characterization of the environmental conditions that define the climatic niche can be used to improve the delimitation of T. pallidipennis haplogroups that constitute cryptic species.

Molecular data confirm Triatoma pallidipennis Stål, 1872 (Hemiptera: Reduviidae: Triatominae) as a novel cryptic species complex.

Triatoma pallidipennis constitues one of the most important Chagas disease vector in Mexico. Previous studies based on molecular data suggest T. pallidipennis as a complex of cryptic species. For that reason, we analyzed the phylogenetic relationships of T. pallidipennis using DNA sequences from the mitochondrial ND4 gene and the ITS-2 gene. In addition, the divergence times were estimated, and possible new taxa were delimited with three species delimitation methods. Finally, genetic distances and possible connectivity routes based on shared haplotypes were obtained among the T. pallidipennis populations. Five haplogroups (possible cryptic species) were found, based on delimitation methods and genetic distances. Haplogroup divergence began about 3 Ma, in the Pleistocene. Moreover, none of the haplogroups showed potential connectivity routes between them, evidencing lack of gene flow. Our results suggest the existence of a new cryptic species complex within what is currently recognized as a T. pallidipennis.

Molecular systematics of the Reithrodontomys tenuirostris group (Rodentia: Cricetidae) highlighting the Reithrodontomys microdon species complex.

The Reithrodontomys tenuirostris species group is considered "the most specialized" within the genus Reithrodontomys from morphological and ecological perspectives. Previous studies based on molecular data recommended changes in the taxonomy of the group. In particular, R. microdon has been the most taxonomically questioned, with the suggestion that it constitutes a complex of cryptic species. We analyzed the phylogenetic relationships of the R. tenuirostris species group using DNA sequences from the mitochondrial Cytochrome b gene and Intron 7 of the nuclear beta fibrinogen gene. In addition, divergence times were estimated, and possible new taxa delimited with three widely used species delimitation methods. Finally, possible connectivity routes based on shared haplotypes were tested among the R. microdon populations. All species were recovered as monophyletic with the exception of R. microdon, whose individuals were grouped into four different haplogroups, one of which included specimens of R. bakeri. Diversification within the R. tenuirostris species group began about 3 Ma, in the Pleistocene. The bGMYC and STACEY delimitation methods were congruent with each other, delimiting at the species-level each haplogroup within R. microdon, while the mPTP suggested a greater number of species. Moreover, none of the haplogroups showed potential connectivity routes between them, evidencing lack of gene flow. Our results suggest the existence of a higher number of species in the R. tenuirostris group, because we show that there are four species within what is currently recognized as R. microdon.

Dentro del género Reithrodontomys, el grupo de especies R. tenuirostris es considerado "el más especializado" morfológica y ecológicamente. Estudios moleculares previos recomendaron cambios en su taxonomía, proponiendo a R. microdon como un complejo de especies crípticas. Se analizaron las relaciones filogenéticas del grupo de especies R. tenuirostris con base en información de un gen mitocondrial, Citocromo b, y uno nuclear, el intrón 7 del beta fibrinógeno. Se estimaron los tiempos de divergencia, y se delimitó a posibles nuevos taxa aplicando tres métodos comúnmente utilizados, y se evaluaron posibles rutas de conectividad con base en los diferentes haplotipos identificados en las poblaciones de R. microdon. Todas las especies del grupo se recuperaron como monofiléticas excepto R. microdon, cuyos individuos formaron cuatro haplogrupos diferentes, uno de los cuales incluyó a especímenes de R. bakeri. Según la datación obtenida, la diversificación del grupo R. tenuirostris comenzó en el Pleistoceno, hace aproximadamente 3 Ma. Los métodos de análisis de delimitación de especies bGMYC y STACEY resultaron congruentes entre sí, logrando delimitar cada haplogrupo dentro de R. microdon a nivel de especie, mientras que con el método de mPTP se delimitó un número mayor de especies. No se identificaron rutas de conectividad entre haplogrupos, lo que resultaría en una ausencia de flujo génico. Se sugiere la existencia de un mayor número de especies en el grupo R. tenuirostris, ya que hay cuatro especies incluidas en lo que actualmente se reconoce como R. microdon.

Quantitative imagery analysis of spot patterns for the three-haplogroup classification of Triatoma dimidiata (Latreille, 1811) (Hemiptera: Reduviidae), an important vector of Chagas disease.

BACKGROUND: Spots and coloring patterns evaluated quantitatively can be used to discriminate and identify possible cryptic species. Species included in the Triatoma dimidiata (Reduviidae: Triatominae) complex are major disease vectors of Chagas disease. Phylogenetic studies have defined three haplogroups for Mexico and part of Central America. We report here our evaluation of the possibility of correctly discriminating these three T. dimidiata haplogroups using the pattern of the dorsal spots. METHODS: Digital images of the dorsal region of individuals from the three haplogroups were used. Image processing was used to extract primary and secondary variables characterizing the dorsal spot pattern. Statistical analysis of the variables included descriptive statistics, non-parametric Kruskal-Wallis tests, discriminant function analysis (DFA) and a neural classification network. RESULTS: A distinctive spot pattern was found for each haplogroup. The most differentiated pattern was presented by haplogroup 2, which was characterized by its notably larger central spots. Haplogroups 1 and 3 were more similar to each other, but there were consistent differences in the shape and orientation of the spots. Significant differences were found among haplogroups in almost all of the variables analyzed, with the largest differences seen for relative spot area, mean relative area of central spots, central spots Feret diameter and lateral spots Feret diameter and aspect ratio. Both the DFA and the neural network had correct discrimination values of > 90%. CONCLUSIONS: Based on the results of this analysis, we conclude that the spot pattern can be reliably used to discriminate among the three haplogroups of T. dimidiata in Mexico, and possibly among triatomine species.

Identifying Chagas disease vectors using elliptic Fourier descriptors of body contour: a case for the cryptic dimidiata complex.

BACKGROUND: Triatoma dimidiata (Reduviidae: Triatominae) is an important vector of Chagas disease in various countries in the Americas. Phylogenetic studies have defined three lineages in Mexico and part of Central America. While there is a marked genetic differentiation, methods for identifying them using morphometric analyses with landmarks have not yet been fully resolutive. Elliptical Fourier descriptors (EFDs), which mathematically describe the shape of any closed two-dimensional contours, could be a potentially useful alternative method. Our objective was to validate the use of EFDs for the identification of three lineages of this species complex. METHOD: A total of 84 dorsal view images of individuals of the three lineages were used. Body contours were described with EFDs using between 5 and 30 harmonics. The number of obtained coefficients was reduced by a principal components analysis and the first axis scores were used as shape variables. A linear discriminant function analysis and an ordination plot of the discriminant analysis were performed using the shape variables. A confusion matrix of the ordination plot of the discriminant analysis was obtained to estimate the classification errors, the first five PC scores were statistically compared, and a neural network were then performed using the shape variables. RESULTS: The first principal component explained 50% of the variability, regardless the number of harmonics used. The results of discriminant analysis get improved by increasing the number of harmonics and components considered. With 25 harmonics and 30 components, the identification of haplogroups was achieved with an overall efficiency greater than 97%. The ordering diagram showed the correct discrimination of haplogroups, with only one error of discrimination corroborated by the confusion matrix. When comparing the first five PC scores, significant differences were found among at least two haplogroups. The 30 multilayer perceptron neural networks were also efficient in identification, reaching 91% efficiency with the validation data. CONCLUSIONS: The use of EFD is a simple and useful method for the identification of the main lineages of Triatoma dimidiata, with high values of correct identification.

An improved and low-cost protocol for high-quality DNA isolation for the Chagas disease vectors.

An improved protocol for DNA extraction for the Chagas disease vectors is proposed based on modification to a low cost method described twenty years ago. Quality DNA and high molecular weight were obtained from all samples. NADH-4 gene was successfully amplified by PCR using the isolated DNA. The extraction protocol presented in this technical note is a fast, low-cost, and non-aggressive method to human health for obtaining genetic data from this group of epidemiological importance and potentially other insects.

Species Identity Supersedes the Dilution Effect Concerning Hantavirus Prevalence at Sites across Texas and México.

Recent models suggest a relationship exists between community diversity and pathogen prevalence, the proportion of individuals in a population that are infected by a pathogen, with most inferences tied to assemblage structure. Two contrasting outcomes of this relationship have been proposed: the "dilution effect" and the "amplification effect." Small mammal assemblage structure in disturbed habitats often differs from assemblages in sylvan environments, and hantavirus prevalence is often negatively correlated with habitats containing high species diversity via dilution effect dynamics. As species richness increases, prevalence of infection often is decreased. However, anthropogenic changes to sylvan landscapes have been shown to decrease species richness and/or increase phylogenetic similarities within assemblages. Between January 2011 and January 2016, we captured and tested 2406 individual small mammals for hantavirus antibodies at 20 sites across Texas and México and compared differences in hantavirus seroprevalence, species composition, and assemblage structure between sylvan and disturbed habitats. We found 313 small mammals positive for antibodies against hantaviruses, evincing an overall prevalence of 9.7% across all sites. In total, 40 species of small mammals were identified comprising 2 taxonomic orders (Rodentia and Eulipotyphla). By sampling both habitat types concurrently, we were able to make real-world inferences into the efficacy of dilution effect theory in terms of hantavirus ecology. Our hypothesis predicting greater species richness higher in sylvan habitats compared to disturbed areas was not supported, suggesting the characteristics of assemblage structure do not adhere to current conceptions of species richness negatively influencing prevalence via a dilution effect.

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

BACKGROUND: Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. RESULTS: Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. CONCLUSIONS: We present simple and efficient methodologies for protoplast isolation from multiple P. vulgaris tissues. We also provide a high-efficiency and amenable method for leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT leaf disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools for P. vulgaris research.

Molecular phylogenetics and phylogeographic structure of Sumichrast's harvest mouse (Reithrodontomys sumichrasti: Cricetidae) based on mitochondrial and nuclear DNA sequences.

Sumichrast's harvest mouse (Reithrodontomys sumichrasti) is a montane rodent species widely distributed through the Mesoamerican highlands. We used sequence data from one mitochondrial (cytochrome b) and two nuclear (ß-fibrinogen and acid phosphatase type V) genes for a total of 1962 base pairs to estimate genealogical relationships and assess population genetic structure across the range of this taxon. Maximum likelihood and Bayesian approaches using cytochrome b resolved several major clades, revealing considerably more genetic diversity than observed in previous studies. The basal split in the tree topologies corresponded to the geographical separation among samples on either side of the Isthmus of Tehuantepec in México. We estimated an early Pleistocene or late Pliocene divergence between these two groups. We also recovered a well-supported clade south of the Nicaraguan Depression in Central America that we consider a separate biological species. The 12 networks generated using statistical parsimony (TCS) for cytochrome b sequence data were largely concordant with the phylogenetic analyses and we document the co-occurrence of two of these networks in central Veracurz. Phylogenies derived from ß-fibrinogen and acid phosphatase type V gene segments revealed less phylogenetic signal and did not separate samples of R. sumichrasti east and west of the Isthmus of Tehuantepec. The phylogeny estimated by combining the mitochondrial and nuclear sequence data was essentially identical to the cytochrome b gene tree.

Antibodies to Tacaribe serocomplex viruses (family Arenaviridae, genus Arenavirus) in cricetid rodents from New Mexico, Texas, and Mexico.

Blood samples from 4893 cricetid rodents were tested for antibody (immunoglobulin G) to Whitewater Arroyo virus and Amaparí virus to extend our knowledge of the natural host range and geographical distribution of Tacaribe serocomplex viruses in North America. Antibodies to arenaviruses were found in northern pygmy mice (Baiomys taylori), woodrats (Neotoma spp.), northern grasshopper mice (Onychomys leucogaster), oryzomys (Oryzomys spp.), deermice (Megadontomys nelsoni and Peromyscus spp.), harvest mice (Reithrodontomys spp.), and cotton rats (Sigmodon spp.) captured in New Mexico, Texas, or Mexico. Comparison of endpoint antibody titers to Whitewater Arroyo virus and Amaparí virus in individual blood samples indicated that the Tacaribe complex viruses enzootic in Texas and Mexico are antigenically diverse.

Molecular systematics of Middle American harvest mice Reithrodontomys (Muridae), estimated from mitochondrial cytochrome b gene sequences.

We estimated phylogenetic relationships among 16 species of harvest mice using sequences from the mitochondrial cytochrome b (cyt b) gene. Gene phylogenies constructed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) optimality criteria were largely congruent and arranged taxa into two groups corresponding to the two recognized subgenera (Aporodon and Reithrodontomys). All analyses also recovered R. mexicanus and R. microdon as polyphyletic, although greater resolution was obtained using ML and BI approaches. Within R. mexicanus, three clades were identified with high nodal support (MP and ML bootstrap, Bremer decay and Bayesian posterior probabilities). One represented a subspecies of R. mexicanus from Costa Rica (R. m. cherrii) and a second was distributed in the Sierra Madre Oriental of Mexico. The third R. mexicanus clade consisted of mice from southern Mexico southward to South America. Polyphyly between the two moieties of R. microdon corresponded to the Isthmus of Tehuantepec in southern Mexico. Populations of R. microdon microdon to the east of the isthmus (Chiapas, Mexico) grouped with R. tenuirostris, whereas samples of R. m. albilabris to the west in Oaxaca, Mexico, formed a clade with R. bakeri. Within the subgenus Reithrodontomys, all analyses recovered R. montanus and R. raviventris as sister taxa, a finding consistent with earlier studies based on allozymes and cyt b data. There was also strong support (ML and BI criteria) for a clade consisting of ((R. megalotis, R. zacatecae) (R. sumichrasti)). In addition, cytb gene phylogenies (MP, ML, and BI) recovered R. fulvescens and R. hirsutus (ML and BI) as basal taxa within the subgenus Reithrodontomys. Constraint analyses demonstrated that tree topologies treating the two subgenera (Aporodon and Reithrodontomys) as monophyletic (ML criterion) was significantly better (p>0.036) and supported polyphyly of R. mexicanus (both ML and MP criteria - p>0.013) and R. microdon (MP criterion only for certain topologies; p>0.02). Although several species-level taxa were identified based on multiple, independent data sets, we recommended a conservative approach which will involve thorough analyses of museum specimens including material from type localities together with additional sampling and data from multiple, nuclear gene markers.

Comparative Phylogeography of Mesoamerican Highland Rodents: Concerted versus Independent Response to Past Climatic Fluctuations.

The phylogeography of Sumichrast's harvest mouse (Reithrodontomys sumichrasti) was examined through maximum-likelihood and parsimony analyses of 1,130 bp of mitochondrial Cytochrome b sequence data from 43 individuals. The phylogeography of this Middle American highland forest-dwelling species was compared to that previously published for the codistributed Aztec deer mouse complex (Peromyscus aztecus/Peromyscus hylocetes complex) in order to test competing hypotheses of concerted versus independent responses of codistributed forms to past climatic fluctuations. Qualitatively, there were strong similarities in the phylogeographic patterns of the two groups, yet there were also areas of incongruence. Likelihood-ratio tests (Kishino-Hasegawa-Templeton and parametric bootstrap tests) indicated that this incongruence is significant and cannot be attributed simply to uncertainty in phylogenetic estimation, thereby falsifying the concerted-response hypothesis. Conversely, tree-reconciliation analysis of the area relationships inferred for each group separately indicated that there has been a significant history of covicariance between the two groups, falsifying the independent-response hypothesis. It appears that codistributed taxa in the geologically complex highlands of Mesoamerica share more common biogeographical history than can be accounted for by the independent-response hypothesis yet have not responded to past climatic fluctuations in the lock-step fashion predicted by the concerted-response hypothesis.