Revisiting gene typing and phylogeny of Trypanosoma cruzi reference strains: Comparison of the relevance of mitochondrial DNA, single-copy nuclear DNA, and the intergenic region of mini-exon gene.

Chagas disease is a widespread neglected disease in Latin America. Trypanosoma cruzi, the causative agent of the disease, is currently subdivided into six DTUs (discrete typing units) named TcI-TcVI, and although no clear association has been found between parasite genetics and different clinical outcomes of the disease or different transmission cycles, genetic characterization of T. cruzi strains remains crucial for integrated epidemiological studies. Numerous markers have been used for this purpose, although without consensus. These include mitochondrial genes, single or multiple-copy nuclear genes, ribosomal RNA genes, and the intergenic region of the repeated mini-exon gene. To increase our knowledge of these gene sequences and their usefulness for strain typing, we sequenced fragments of three mitochondrial genes, nine single-copy nuclear genes, and the repeated intergenic part of the mini-exon gene by Next Generation Sequencing (NGS) on a sample constituted of 16 strains representative of T. cruzi genetic diversity, to which we added the corresponding genetic data of the 38 T. cruzi genomes fully sequenced until 2022. Our results show that single-copy nuclear genes remain the gold standard for characterizing T. cruzi strains; the phylogenetic tree from concatenated genes (3959 bp) confirms the six DTUs previously recognized and provides additional information about the alleles present in the hybrid strains. In the tree built from the three mitochondrial concatenated genes (1274 bp), three main clusters are identified, including one with TcIII, TcIV, TcV, and TcVI DTUs which are not separated. Nevertheless, mitochondrial markers remain necessary for detecting introgression and heteroplasmy. The phylogenetic tree built from the sequence alignment of the repeated mini-exon gene fragment (327 bp) displayed six clusters, but only TcI was associated with a single cluster. The sequences obtained from strains belonging to the other DTUs were scattered into different clusters. Therefore, while the mini-exon marker may bring, for some biological samples, some advantages in terms of sensibility due to its repeated nature, mini-exon sequences must be used with caution and, when possible, avoided for T. cruzi typing and phylogenetic studies.

Rhodnius (Stål, 1859) (Hemiptera, Triatominae) genus in Bolivian Amazonia: a risk for human populations?

BACKGROUND: Chagas disease, one of the most important neglected tropical diseases in the countries of Latin America, is considered to be a particularly important public health concern in the Amazon region due to increases in the number of outbreaks of acute Chagas disease and increased local transmission in the last 20 years. However, relative to other countries, in Bolivia there is little information available on its transmission in the Amazon region. The aim of this study was to investigate the infestation of palm trees, the main habitat of Triatominae in the region, in several localities, to evaluate the danger they represent to inhabitants. METHODS: Triatominae were collected using live bait traps left overnight in six localities in Pando and Beni Departments, Bolivia. DNA extraction and sequencing were used to establish the Triatominae species (Cytb, 16S and 28S-D2 gene fragments), and the blood meal sources (Cytb fragment). Trypanosoma sp. infection was analyzed by sequencing gene fragments (GPX, GPI, HMCOAR, LAP, PDH and COII) or by mini-exon multiplex PCR. RESULTS: A total of 325 Rhodnius were captured (97.3% of nymphs) from the 1200 traps placed in 238 palm trees and 32 burrows/ground holes. Sequence analyses on DNA extracted from 114 insects and phylogeny analysis identified two triatomine species: Rhodnius stali (17%) and Rhodnius montenegrensis (equated to Rhodnius robustus II, 83%). These were found in palm trees of the genera Attalea (69%), Astrocaryum (13%), Copernicia (12%), Euterpe (2%) and Acrocomia (1%). The infection rate was around 30% (165 analyzed insects), with 90% of analyzed insects infected by Trypanosoma cruzi (only the TcI discrete typing unit was detected), 3% infected by Trypanosoma rangeli (first time found in Bolivian Triatominae) and 7% infected by mixed T. cruzi (TcI)-T. rangeli. Rhodnius specimens fed on Didelphidae, rodents, gecko and humans. CONCLUSIONS: The results of this study highlight the epidemiological importance of Rhodnius in the Bolivian Amazon region. The huge geographical distribution of Rhodnius and their proximity to the human dwellings, high infection rate and frequent meals on the human population highlight a risk of transmission of Chagas disease in the region.

Molecular data supports monophyly of Triatoma dispar complex within genus Triatoma.

The genus Triatoma contains numerous species, principal or secondary vectors of Chagas disease, which have been included in the three main lineages of Triatomini tribe based on morphological and biogeographical characteristics: North American, South American, and T. dispar complex. The three members of the T. dispar complex are distributed in Ecuador. This complex has been scarcely studied through molecular approaches, and the taxonomic position of this complex is not confirmed. In this study, we explored the phylogenetic relationships within the genus Triatoma, including five species from North and Central America, six from South America, and the three species belonging to the T. dispar complex. Partial sequences of four mitochondrial genes (Cyt b, COII, 16S-rRNA, 12S-rRNA) and two nuclear genes (18S-rRNA, ITS2) were obtained from 74 specimens. Phylogenetic trees were built with concatenated and single sequences through maximum likelihood (ML), maximum parsimony (MP), and Bayesian methods. The trees built using concatenated sequences showed three main branches (clusters) highly supported by significant bootstrap values; the T. dispar complex appeared as a monophyletic group separate from species of North and Central American origin and South American origin. On the contrary, for each gene tree, the three main clusters were not always significantly supported, mostly because genetic information is dramatically reduced when a single gene is considered. Consequently, concatenation of genes gives relevant results and is highly recommended for further in-depth examination of the relationships of several species and complexes of triatomines that remain unresolved. Moreover, our current molecular data fully revealed the division of genus Triatoma into at least three main genetic groups.

Chagas vectors Panstrongylus chinai (Del Ponte, 1929) and Panstrongylus howardi (Neiva, 1911): chromatic forms or true species?

BACKGROUND: Chagas disease is a parasitic infection transmitted by "kissing bugs" (Hemiptera: Reduviidae: Triatominae) that has a huge economic impact in Latin American countries. The vector species with the upmost epidemiological importance in Ecuador are Rhodnius ecuadoriensis (Lent & Leon, 1958) and Triatoma dimidiata (Latreille, 1811). However, other species such as Panstrongylus howardi (Neiva, 1911) and Panstrongylus chinai (Del Ponte, 1929) act as secondary vectors due to their growing adaptation to domestic structures and their ability to transmit the parasite to humans. The latter two taxa are distributed in two different regions, they are allopatric and differ mainly by their general color. Their relative morphological similarity led some authors to suspect that P. chinai is a melanic form of P. howardi. METHODS: The present study explored this question using different approaches: antennal phenotype; geometric morphometrics of heads, wings and eggs; cytogenetics; molecular genetics; experimental crosses; and ecological niche modeling. RESULTS: The antennal morphology, geometric morphometrics of head and wing shape and cytogenetic analysis were unable to show distinct differences between the two taxa. However, geometric morphometrics of the eggs, molecular genetics, ecological niche modeling and experimental crosses including chromosomal analyses of the F1 hybrids, in addition to their coloration and current distribution support the hypothesis that P. chinai and P. howardi are separate species. CONCLUSIONS: Based on the evidence provided here, P. howardi and P. chinai should not be synonymized. They represent two valid, closely related species.

Genetic data support speciation between Panstrongylus howardi and Panstrongylus chinai, vectors of Chagas disease in Ecuador.

Limited genetic data are currently available for three vectors of Chagas disease in Ecuador, Panstrongylus howardi, P. chinai, and P. rufotuberculatus. Previously regarded as mainly sylvatic, these species have been poorly studied. Recently, they have been more frequently reported in domiciles and peridomiciles and are now considered true secondary vectors of Chagas disease in a country where an estimated 200,000 people are infected by Trypanosoma cruzi, a causative agent of this disease. In order to fill this gap, we obtained DNA for sequencing from 53 insects belonging to these three species and mainly sampled from the two Ecuadorian provinces of Loja and Manabí. We used six mitochondrial loci (COI, COII, ND4, CytB, 16S, and 12S) and two nuclear ones (ITS2 and 18S). We interpreted the phylogenetic trees built with single and concatenated data through maximum likelihood, Bayesian Markov chain Monte Carlo, and maximum parsimony methods. We provide evidence that P. chinai and P. howardi are indeed two supported species closely related and derived from a common ancestor. Additionally, the phylogenetic position of P. rufotuberculatus was confirmed as being distant from P. chinai and P. howardi and clustered with Triatoma dimidiata, a species belonging to the Northern American Triatoma clade.

Triatominae: does the shape change of non-viable eggs compromise species recognition?

BACKGROUND: Eggs have epidemiological and taxonomic importance in the subfamily Triatominae, which contains Chagas disease vectors. The metric properties (size and shape) of eggs are useful for distinguishing between close species, or different geographical populations of the same species. METHODS: We examined the effects of egg viability on its metric properties, and the possible consequences on species recognition. Four species were considered: Panstrongylus chinai, P. howardi and Triatoma carrioni (tribe Triatomini), and Rhodnius ecuadoriensis (tribe Rhodniini). Digitization was performed on pictures taken when the viability of the egg could not clearly be predicted by visual inspection. We then followed development to separate viable from non-viable eggs, and the metric changes associated with viability status of the eggs were tested for species discrimination (interspecific difference). RESULTS: The shape of the complete contour of the egg provided satisfactory species classification (95% of correct assignments, on average), with improved scores (98%) when discarding non-viable eggs from the comparisons. Using only non-viable eggs, the scores dropped to 90%. The morphometric differences between viable and non-viable eggs were also explored (intraspecific comparison). A constant metric change observed was a larger variance of size and shape in non-viable eggs. For all species, larger eggs, or eggs with larger operculum, were more frequently non-viable. However, these differences did not allow for an accurate prediction regarding egg viability. CONCLUSIONS: The strong taxonomic signal present in egg morphology was affected by the level of viability of the eggs. The metric properties as modified in non-viable eggs presented some general trends which could suggest the existence of an optimum phenotype for size and for shape. Globally, viable eggs tended to have intermediate or small sizes, and presented a less globular shape in the Triatomini, or a relatively wider neck in Rhodnius ecuadoriensis.

The modern morphometric approach to identify eggs of Triatominae.

BACKGROUND: Egg morphometrics in the Triatominae has proved to be informative for distinguishing tribes or genera, and has been based generally on traditional morphometrics. However, more resolution is required, allowing species or even population recognition, because the presence of eggs in the domicile could be related to the species ability to colonize human dwellings, suggesting its importance as a vector. RESULTS: We explored the resolution of modern morphometric methods to distinguish not only tribes and genera, but also species or geographic populations in some important Triatominae. Four species were considered, representing two tribes and three genera: Panstrongylus chinai and P. howardi, Triatoma carrioni and Rhodnius ecuadoriensis. Within R. ecuadoriensis, two geographical populations of Ecuador were compared. For these comparisons, we selected the most suitable day of egg development, as well as the possible best position of the egg for data capture. The shape of the eggs in the Triatominae does not offer true anatomical landmarks as the ones used in landmark-based morphometrics, except for the egg cap, especially in eggs with an evident "neck", such as those of the Rhodniini. To capture the operculum shape variation, we used the landmark- and semilandmark-based method. The results obtained from the metric properties of the operculum were compared with the ones provided by the simple contour of the whole egg, as analyzed by the Elliptic Fourier Analysis. Clear differences could be disclosed between the genera, between the species - among which two very close species (P. chinai and P. howardi), as well as between two allopatric, conspecific populations. The whole egg contour (including the operculum) produced reclassification scores much more satisfactory than the ones obtained using the operculum only. CONCLUSIONS: We propose the outline-based approach as the most convenient characterization tool to identify unknown eggs at the species or population levels.

Un tejido de sueños, la conformación de una red, unión de voluntades y algo más: sistematización de la experiencia de conformación de la red de salud sexual y reproductiva: Aportes para la reflexión / A Textile of dreams

Presenta el fortalecimiento de procesos de concertación y participación a nivel local para mejorar el acceso a los servicios de salud sexual y reproductiva de las poblaciones pobres, en este caso a través de la conformación de la Red de Salud Sexual y Reproductiva...