Comparative proteomic analysis of the hemolymph and salivary glands of Rhodnius prolixus and R. colombiensis reveals candidates associated with differential lytic activity against Trypanosoma cruzi Dm28c and T. cruzi Y.

BACKGROUND: Immune response of triatomines plays an important role in the success or failure of transmission of T. cruzi. Studies on parasite-vector interaction have shown the presence of trypanolytic factors and have been observed to be differentially expressed among triatomines, which affects the transmission of some T. cruzi strains or DTUs (Discrete Typing Units). METHODOLOGY/PRINCIPAL FINDINGS: Trypanolytic factors were detected in the hemolymph and saliva of R. prolixus against epimastigotes and trypomastigotes of the Y strain (T. cruzi II). To identify the components of the immune response that could be involved in this lytic activity, a comparative proteomic analysis was carried out, detecting 120 proteins in the hemolymph of R. prolixus and 107 in R. colombiensis. In salivary glands, 1103 proteins were detected in R. prolixus and 853 in R. colombiensis. A higher relative abundance of lysozyme, prolixin, nitrophorins, and serpin as immune response proteins was detected in the hemolymph of R. prolixus. Among the R. prolixus salivary proteins, a higher relative abundance of nitrophorins, lipocalins, and triabins was detected. The higher relative abundance of these immune factors in R. prolixus supports their participation in the lytic activity on Y strain (T. cruzi II), but not on Dm28c (T. cruzi I), which is resistant to lysis by hemolymph and salivary proteins of R. prolixus due to mechanisms of evading oxidative stress caused by immune factors. CONCLUSIONS/SIGNIFICANCE: The lysis resistance observed in the Dm28c strain would be occurring at the DTU I level. T. cruzi I is the DTU with the greatest geographic distribution, from the south of the United States to central Chile and Argentina, a distribution that could be related to resistance to oxidative stress from vectors. Likewise, we can say that lysis against strain Y could occur at the level of DTU II and could be a determinant of the vector inability of these species to transmit T. cruzi II. Future proteomic and transcriptomic studies on vectors and the interactions of the intestinal microbiota with parasites will help to confirm the determinants of successful or failed vector transmission of T. cruzi DTUs in different parts of the Western Hemisphere.

High-throughput analysis of the Trypanosoma cruzi minicirculome (mcDNA) unveils structural variation and functional diversity.

Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia's diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed.

Enhancing Trypanosomatid Identification and Genotyping with Oxford Nanopore Sequencing: Development and Validation of an 18S rRNA Amplicon-Based Method.

Trypanosomatids, including Trypanosoma and Leishmania species, present significant medical and veterinary challenges, causing substantial economic losses, health complications, and even fatalities. Diagnosing and genotyping these species and their genotypes is often complex, involving multiple steps. This study aimed to develop an amplicon-based sequencing (ABS) method using Oxford Nanopore long-read sequencing to enhance Trypanosomatid detection and genotyping. The 18S rDNA gene was targeted for its inter-species conservation. The Trypanosomatid-ABS method effectively distinguished between 11 Trypanosoma species (including Trypanosoma evansi, Trypanosoma theileri, Trypanosoma vivax, and Trypanosoma rangeli) and 6 Trypanosoma cruzi discrete typing units (TcI to TcVI and TcBat), showing strong concordance with conventional methods (κ index of 0.729, P < 0.001). It detected co-infections between Trypanosomatid genera and T. cruzi, with a limit of detection of one parasite per mL. The method was successfully applied to human, animal, and triatomine samples. Notably, TcI predominated in chronic Chagas samples, whereas TcII and TcIV were found in the acute stage. Triatomine vectors exhibited diverse Trypanosomatid infections, with Triatoma dimidiata mainly infected with TcI and occasional TcBat co-infections, and Rhodnius prolixus showing TcI and TcII infections, along with T. rangeli co-infections and mixed TcII infections. Animals were infected with T. vivax, T. theileri, and T. evansi. The ABS method's high resolution, sensitivity, and accuracy make it a valuable tool for understanding Trypanosomatid dynamics, enhancing disease control strategies, and enabling targeted interventions.

A phased genome assembly of a Colombian Trypanosoma cruzi TcI strain and the evolution of gene families.

Chagas is an endemic disease in tropical regions of Latin America, caused by the parasite Trypanosoma cruzi. High intraspecies variability and genome complexity have been challenges to assemble high quality genomes needed for studies in evolution, population genomics, diagnosis and drug development. Here we present a chromosome-level phased assembly of a TcI T. cruzi strain (Dm25). While 29 chromosomes show a large collinearity with the assembly of the Brazil A4 strain, three chromosomes show both large heterozygosity and large divergence, compared to previous assemblies of TcI T. cruzi strains. Nucleotide and protein evolution statistics indicate that T. cruzi Marinkellei separated before the diversification of T. cruzi in the known DTUs. Interchromosomal paralogs of dispersed gene families and histones appeared before but at the same time have a more strict purifying selection, compared to other repeat families. Previously unreported large tandem arrays of protein kinases and histones were identified in this assembly. Over one million variants obtained from Illumina reads aligned to the primary assembly clearly separate the main DTUs. We expect that this new assembly will be a valuable resource for further studies on evolution and functional genomics of Trypanosomatids.

Distribución de Aedes albopictus en Ibagué: potencial riesgo de brotes de arbovirosis

Introducción. Aedes albopictus es un vector de arbovirus como dengue, Zika, chikungunya y fiebre amarilla. Los primeros reportes en el continente americano datan de 1985 y dada su capacidad de adaptación ecológica y fisiológica, se ha distribuido rápidamente en el territorio colombiano desde su primer reporte en 1998. Objetivo. Determinar la distribución de A. albopictus en las comunas de Ibagué, Colombia. Materiales y métodos. Los muestreos se realizaron entre mayo y noviembre de 2022 en zonas con abundante vegetación de las 13 comunas de Ibagué. Se emplearon aspiradores y redes entomológicas. Los mosquitos fueron transportados al Laboratorio de Investigaciones en Parasitología Tropical de la Universidad del Tolima para su determinación taxonómica. Resultados. Se identificaron 708 ejemplares de A. albopictus, distribuidos en las comunas de Ibagué. La mayor abundancia del vector se presentó en las comunas 10, 11, 7, 8, 2 y 9. Las comunas 3, 4, 5, 6, 12 y 13 presentaron abundancias relativas cercanas al 3 %, y la comuna 1 tuvo una abundancia del 2 %. Conclusiones. Aedes albopictus está distribuido en todas las comunas de Ibagué, probablemente su dispersión se ha visto favorecida por las condiciones ambientales y sociales de esta región. Se recomienda hacer seguimiento anual a las poblaciones de este vector y realizar una caracterización molecular de los arbovirus encontrados. Además, el conocer la distribución de este mosquito en la ciudad permitirá focalizar las estrategias de control entomológico y prevenir futuros brotes de arbovirosis.

Introduction. Aedes albopictus is a vector for arboviruses, such as dengue, Zika, chikungunya, and yellow fever. The first A. albopictus reports on the American continent date back to 1985. It has spread rapidly throughout Colombia since its first report in 1998 due to its ecological and physiological adaptation capability. Objective. To determine A. albopictus distribution in the 13 communes of Ibagué, Colombia. Materials and methods. Samples were collected between May and November 2022 in the 13 communes of Ibagué. Vacuum sampling and sweep-netting entomological nets were used in areas with abundant vegetation. The mosquitoes were transported to the Laboratorio de Investigaciones en Parasitología Tropical at the Universidad del Tolima for taxonomic determination. Results. We identified 708 A. albopictus specimens distributed throughout Ibague's 13 communes. The highest vector abundance occurred in communes 10, 11, 7, 8, 2, and 9; communes 3, 4, 5, 6, 12, and 13 had a relative abundance of around 3%, while commune 1 had 2% of relative abundance. Conclusions. Aedes albopictus is distributed throughout all the communes of Ibague. Its dispersion has probably been favored by this region's environmental and social conditions. We recommend annual monitoring of these vectors populations and molecular characterization of the found arboviruses. Ascertaining this mosquito's distribution throughout the city will enable focusing entomological control strategies and preventing future arbovirus outbreaks.

Aedes albopictus distribution in Ibagué, Colombia: Potential risk of arboviral outbreaks / Distribución de Aedes albopictus en Ibagué: potencial riesgo de brotes de arbovirosis

Introduction: Aedes albopictus is a vector for arboviruses, such as dengue, Zika, chikungunya, and yellow fever. The first A. albopictus reports on the American continent date back to 1985. It has spread rapidly throughout Colombia since its first report in 1998 due to its ecological and physiological adaptation capability. Objective: To determine A. albopictus distribution in the 13 communes of Ibagué, Colombia. Materials and methods: Samples were collected between May and November 2022 in the 13 communes of Ibagué. Vacuum sampling and sweep-netting entomological nets were used in areas with abundant vegetation. The mosquitoes were transported to the Laboratorio de Investigaciones en Parasitología Tropical at the Universidad del Tolima for taxonomic determination. Results: We identified 708 A. albopictus specimens distributed throughout Ibague's 13 communes. The highest vector abundance occurred in communes 10, 11, 7, 8, 2, and 9; communes 3, 4, 5, 6, 12, and 13 had a relative abundance of around 3%, while commune 1 had 2% of relative abundance. Conclusions: Aedes albopictus is distributed throughout all the communes of Ibague. Its dispersion has probably been favored by this region's environmental and social conditions. We recommend annual monitoring of these vectors populations and molecular characterization of the found arboviruses. Ascertaining this mosquito's distribution throughout the city will enable focusing entomological control strategies and preventing future arbovirus outbreaks.

Introducción: Aedes albopictus es un vector de arbovirus como dengue, Zika, chikungunya y fiebre amarilla. Los primeros reportes en el continente americano datan de 1985 y dada su capacidad de adaptación ecológica y fisiológica, se ha distribuido rápidamente en el territorio colombiano desde su primer reporte en 1998. OBJETIVO: Determinar la distribución de A. albopictus en las comunas de Ibagué, Colombia. Materiales y métodos: Los muestreos se realizaron entre  mayo y noviembre de 2022 en zonas con abundante  vegetación de las 13 comunas de Ibagué. Se emplearon aspiradores y redes entomológicas. Los mosquitos fueron transportados al Laboratorio de Investigaciones en  Parasitología Tropical de la Universidad del Tolima para su determinación taxonómica. RESULTADOS: Se identificaron 708 ejemplares de A.  lbopictus, distribuidos en las comunas de Ibagué. La mayor abundancia del vector se presentó en las comunas 10, 11, 7, 8, 2 y 9. Las comunas 3, 4, 5, 6, 12 y 13 presentaron abundancias relativas cercanas al 3 %, y la comuna 1 tuvo una abundancia del 2 %. CONCLUSIONES: Aedes albopictus está distribuido en todas las comunas de Ibagué, probablemente su dispersión se ha visto favorecida por las condiciones ambientales y sociales de esta región. Se recomienda hacer seguimiento anual a las poblaciones de este vector y realizar una caracterización molecular de los arbovirus encontrados. Además, el conocer la distribución de este mosquito en la ciudad permitirá focalizar las estrategias de control entomológico y prevenir futuros brotes de arbovirosis.

Trypanosoma cruzi Parasite Burdens of Several Triatomine Species in Colombia.

Trypanosoma cruzi, the causal agent of Chagas disease, is mainly transmitted by insects of the Triatominae subfamily. In Colombia, there are 26 triatomine species, and 16 of them are naturally infected with the parasite. The parasite loads of naturally infected vectors can be significant in targeting specific species that can affect the epidemiology of the disease. Studying their ecology and behavior is vital to understand their role in T. cruzi transmission dynamics. We evaluated the parasite loads of 182 field-collected triatomines corresponding to 10 species in 13 departments across Colombia. We standardized a methodology to quantify T. cruzi DNA in these insects. We obtained a LOD (limit of detection) of 3.05 p-eq/mL. The 82% of triatomines we evaluated were positive for T. cruzi infection, with loads ranging from hundreds to millions of equivalent parasites per milliliter. Panstrongylus geniculatus, Rhodnius prolixus, and Triatoma dimidiata were the species with the highest loads of T. cruzi; however, other species whose role as vectors is still unknown were also found with high loads of parasites. Our results suggest the relevance of secondary species for T. cruzi transmission in Colombia. We hope our data can help improve entomological surveillance and vector control programs in the country and the region.

Genome plasticity driven by aneuploidy and loss of heterozygosity in Trypanosoma cruzi.

Trypanosoma cruzi the causative agent of Chagas disease shows a marked genetic diversity and divided into at least six Discrete Typing Units (DTUs). High intra genetic variability has been observed in the TcI DTU, the most widely distributed DTU, where patterns of genomic diversity can provide information on ecological and evolutionary processes driving parasite population structure and genome organization. Chromosomal aneuploidies and rearrangements across multigene families represent an evidence of T. cruzi genome plasticity. We explored genomic diversity among 18 Colombian T. cruzi I clones and 15 T. cruzi I South American strains. Our results confirm high genomic variability, heterozygosity and presence of a clade compatible with the TcIdom genotype, described for strains from humans in Colombia and Venezuela. TcI showed high structural plasticity across the geographical region studied. Differential events of whole and segmental aneuploidy (SA) along chromosomes even between clones from the same strain were found and corroborated by the depth and allelic frequency. We detected loss of heterozygosity (LOH) events in different chromosomes, however, the size and location of segments under LOH varied between clones. Genes adjacent to breakpoints were evaluated, and retrotransposon hot spot genes flanked the beginning of segmental aneuploidies. Our results suggest that T. cruzi genomes, like those of Leishmania, may have a highly unstable structure and there is now an urgent need to design experiments to explore any potential adaptive role for the plasticity observed.

Molecular detection of Toxoplasma gondii in meat for human consumption in Ibague, Colombia / Detección molecular de Toxoplasma gondii en carnes para consumo humano en Ibagué, Colombia

Introduction: Toxoplasma gondii is a parasite with great zoonotic potential. It can infect a broad range of warm-blooded hosts (including livestock) and causes significant losses in the industry. In humans, it has been described as a pathogen in immunosuppressed people, it affects the fetus development in congenital infections, and is associated with various behavioral disorders in healthy people. Humans can acquire T. gondii by consuming undercooked, contaminated meat. Objective: To determine T. gondii positivity (currently unknown) in meat for human consumption (i.e., beef, chicken, and pork) in the city of Ibague, Colombia. Materials and methods: We used conventional nested PCR and the T. gondii B1 gene sequence as amplification target. We collected samples of meat (N=186) sold in the urban area of Ibagué (62 beef, 62 chicken, and 62 pork samples) and determined the T. gondii positivity percentage for each type of meat. Results: The study found an average of 18.8% positivity for all meat samples, pork having the highest percentage (22.5%; 14/62), followed by beef (19.3%; 12/62) and chicken (14.5%; 9/62). The best-amplified products were sequenced by macrogen and aligned with the B1 gene sequences in GenBank, thereby confirming their identity. Conclusions: This is the first study of T. gondii prevalence in meat for human consumption carried out in the city of Ibagué and the department of Tolima. All three types of meat sampled represent a risk for local human infection

Introducción. Toxoplasma gondii es un parásito con gran potencial zoonótico que puede infectar un amplio rango de huéspedes de sangre caliente, incluidos los animales del sector pecuario, lo que causa pérdidas a la industria. En el humano, es patógeno en personas inmunosuprimidas y afecta el desarrollo del feto en infecciones congénitas. Además, se asocia con diversos trastornos del comportamiento en personas sanas. El humano puede adquirir T. gondii al consumir carnes contaminadas mal cocidas. Objetivo. Determinar la positividad de T. gondii en carnes de consumo humano (res, pollo y cerdo) en Ibagué, Colombia. Materiales y métodos. Se utilizó la PCR convencional anidada y la secuencia del gen B1 de T. gondii como blanco de amplificación. Se tomaron 186 muestras de carne comercializada en la zona urbana de Ibagué (62 de res, 62 de pollo y 62 de cerdo) y se obtuvo el porcentaje de positividad en cada tipo de carne evaluada. Resultados. Se encontró un porcentaje de positividad de 18,8 % en las muestras, siendo la carne de cerdo la del mayor porcentaje (22,5 %; 14/62), seguida por las muestras de carne de res (19,3 %; 12/62) y de pollo (14,5 %; 9/62). Los mejores productos amplificados fueron secuenciados en Macrogen, y alineados con las secuencias del gen B1 depositadas en el GenBank, con lo que se confirmó su identidad. Conclusiones. Este es el primer estudio sobre prevalencia de T. gondii en carnes para consumo humano en Ibagué y el departamento del Tolima. Se demostró que los tres tipos de carne representan un riesgo para la infección en humanos a nivel local.

Detección molecular de Toxoplasma gondii en carnes para consumo humano en Ibagué, Colombia / Molecular detection of Toxoplasma gondii in meat for human consumption in Ibague, Colombia

Introducción. Toxoplasma gondii es un parásito con gran potencial zoonótico que puede infectar un amplio rango de huéspedes de sangre caliente, incluidos los animales del sector pecuario, lo que causa pérdidas a la industria. En el humano, es patógeno en personas inmunosuprimidas y afecta el desarrollo del feto en infecciones congénitas. Además, se asocia con diversos trastornos del comportamiento en personas sanas. El humano puede adquirir T. gondii al consumir carnes contaminadas mal cocidas. Objetivo. Determinar la positividad de T. gondii en carnes de consumo humano (res, pollo y cerdo) en Ibagué, Colombia. Materiales y métodos. Se utilizó la PCR convencional anidada y la secuencia del gen B1 de T. gondii como blanco de amplificación. Se tomaron 186 muestras de carne comercializada en la zona urbana de Ibagué (62 de res, 62 de pollo y 62 de cerdo) y se obtuvo el porcentaje de positividad en cada tipo de carne evaluada. Resultados. Se encontró un porcentaje de positividad de 18,8 % en las muestras, siendo la carne de cerdo la del mayor porcentaje (22,5 %; 14/62), seguida por las muestras de carne de res (19,3 %; 12/62) y de pollo (14,5 %; 9/62). Los mejores productos amplificados fueron secuenciados en Macrogen, y alineados con las secuencias del gen B1 depositadas en el GenBank, con lo que se confirmó su identidad. Conclusiones. Este es el primer estudio sobre prevalencia de T. gondii en carnes para consumo humano en Ibagué y el departamento del Tolima. Se demostró que los tres tipos de carne representan un riesgo para la infección en humanos a nivel local.

Introduction: Toxoplasma gondii is a parasite with great zoonotic potential. It can infect a broad range of warm-blooded hosts (including livestock) and causes significant losses in the industry. In humans, it has been described as a pathogen in immunosuppressed people, it affects the fetus development in congenital infections, and is associated with various behavioral disorders in healthy people. Humans can acquire T. gondii by consuming undercooked, contaminated meat. Objective: To determine T. gondii positivity (currently unknown) in meat for human consumption (i.e., beef, chicken, and pork) in the city of Ibague, Colombia. Materials and methods: We used conventional nested PCR and the T. gondii B1 gene sequence as amplification target. We collected samples of meat (N=186) sold in the urban area of Ibagué (62 beef, 62 chicken, and 62 pork samples) and determined the T. gondii positivity percentage for each type of meat. Results: The study found an average of 18.8% positivity for all meat samples, pork having the highest percentage (22.5%; 14/62), followed by beef (19.3%; 12/62) and chicken (14.5%; 9/62). The best-amplified products were sequenced by macrogen and aligned with the B1 gene sequences in GenBank, thereby confirming their identity. Conclusions: This is the first study of T. gondii prevalence in meat for human consumption carried out in the city of Ibagué and the department of Tolima. All three types of meat sampled represent a risk for local human infection.

Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex.

We sequenced maxicircles from T. cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T. cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T. cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T. cruzi is a complex of species composed by group 1 that contains clades A (TcI), B (TcIII) and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist in T. cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T. cruzi.

Systematic review on the biology, ecology, genetic diversity and parasite transmission potential of Panstrongylus geniculatus (Latreille 1811) in Latin America.

Panstrongylus geniculatus (Latreille, 1811) is the triatomine with the largest geographic distribution in Latin America. It has been reported in 18 countries from southern Mexico to northern Argentina, including the Caribbean islands. Although most reports indicate that P. geniculatus has wild habitats, this species has intrusive habits regarding human dwellings mainly located in intermediate deforested areas. It is attracted by artificial light from urban and rural buildings, raising the risk of transmission of Trypanosoma cruzi. Despite the wide body of published information on P. geniculatus, many knowledge gaps exist about its biology and epidemiological potential. For this reason, we analysed the literature for P. geniculatus in Scopus, PubMed, Scielo, Google Scholar and the BibTriv3.0 databases to update existing knowledge and provide better information on its geographic distribution, life cycle, genetic diversity, evidence of intrusion and domiciliation, vector-related circulating discrete taxonomic units, possible role in oral T. cruzi transmission, and the effect of climate change on its biology and epidemiology.

Systematic review on the biology, ecology, genetic diversity and parasite transmission potential of Panstrongylus geniculatus (Latreille 1811) in Latin America

Panstrongylus geniculatus (Latreille, 1811) is the triatomine with the largest geographic distribution in Latin America. It has been reported in 18 countries from southern Mexico to northern Argentina, including the Caribbean islands. Although most reports indicate that P. geniculatus has wild habitats, this species has intrusive habits regarding human dwellings mainly located in intermediate deforested areas. It is attracted by artificial light from urban and rural buildings, raising the risk of transmission of Trypanosoma cruzi. Despite the wide body of published information on P. geniculatus, many knowledge gaps exist about its biology and epidemiological potential. For this reason, we analysed the literature for P. geniculatus in Scopus, PubMed, Scielo, Google Scholar and the BibTriv3.0 databases to update existing knowledge and provide better information on its geographic distribution, life cycle, genetic diversity, evidence of intrusion and domiciliation, vector-related circulating discrete taxonomic units, possible role in oral T. cruzi transmission, and the effect of climate change on its biology and epidemiology.

Salivary proteins electrophoretic patterns enabled differentiating Colombian Rhodnius Trans-Andean and Cis-Andean groups / [Los patrones electroforéticos de proteínas salivales permiten diferenciar los grupos transandino y cisandino de las especies de Rhodnius de Colombia].

Introduction: Rhodnius (Hemiptera: Reduviidae: Triatominae) species are made up of haematophagous insect vectors for Trypanosoma cruzi (Chagas' disease aetiological agent) and T. rangeli, an infective parasite that is not pathogenic for vertebrate hosts. The study of their salivary protein diversity enables the obtention of characteristic one-dimensional electrophoretic profiles of some triatomine species; however, few reports have dealt with Rhodnius species salivary proteins electrophoretic patterns. Objective: To compare R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus' salivary proteins one-dimensional electrophoretic profiles. Materials and methods: SDS-PAGE was used for obtaining electrophoretic profiles of saliva from the species under study. The unweighted pair group method with arithmetic mean (UPGMA) was used for constructing a phenogram. Results: Electrophoretic profiles of soluble saliva had protein bands ranging from 15 to 45 kDa, thereby enabling the five species studied to be differentiated. The phenogram revealed two main groups, one formed by the Pictipes and Prolixus cis-Andean groups and another consisting of the Pallescens trans-Andean group. Conclusion: Differences were revealed regarding R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus electrophoretic profiles of salivary proteins; their variability facilitated constructing a phenogram which was taxonomically congruent with the groups from the genus Rhodnius.

Introducción. Las especies Rhodnius (Hemiptera: Reduviidae: Triatominae) están conformadas por insectos hematófagos vectores de Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, y T. rangeli, parásito infectivo pero no patógeno para el vertebrado. El estudio de la diversidad proteica de la saliva de estos insectos permite la obtención de perfiles electroforéticos unidimensionales característicos de algunas especies de triatominos. Sin embargo, el reporte de los patrones electroforéticos de proteínas salivales de las especies de Rhodnius ha sido escaso. Objetivo. Hacer un análisis comparativo de los perfiles electroforéticos unidimensionales de las proteínas salivales de R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus. Materiales y métodos. Se obtuvieron los perfiles electroforéticos de la saliva de las especies en estudio mediante electroforesis en gel de poliacrilamida con dodecilsulfatosódico (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) y se construyó un fenograma mediante el método UPGMA (Unweighted Pair Group Method Using Arithmetic Averages). Resultados. Los perfiles electroforéticos de las proteínas solubles de saliva presentaron bandas en un rango de masa aproximado de 15 a 45 kDa, los cuales permitieron diferenciar las cinco especies estudiadas. El fenograma reveló la existencia de dos grupos principales: uno conformado por los grupos cisandinos Pictipes y Prolixus y otro constituido por el grupo transandino Pallescens. Conclusiones. Existen diferencias en los perfiles electroforéticos de las proteínas salivales entre R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus, cuya variabilidad permitió construir un fenograma congruente con los grupos del género Rhodnius.

Transcriptional remodeling during metacyclogenesis in Trypanosoma cruzi I.

Metacyclogenesis is one of the most important processes in the life cycle of Trypanosoma cruzi. In this stage, noninfective epimastigotes become infective metacyclic trypomastigotes. However, the transcriptomic changes that occur during this transformation remain uncertain. Illumina RNA-sequencing of epimastigotes and metacyclic trypomastigotes belonging to T. cruzi DTU I was undertaken. Sequencing reads were aligned and mapped against the reference genome, differentially expressed genes between the two life cycle stages were identified, and metabolic pathways were reconstructed. Gene expression differed significantly between epimastigotes and metacyclic trypomastigotes. The cellular pathways that were mostly downregulated during metacyclogenesis involved glucose energy metabolism (glycolysis, pyruvate metabolism, the Krebs cycle, and oxidative phosphorylation), amino acid metabolism, and DNA replication. By contrast, the processes where an increase in gene expression was observed included those related to autophagy (particularly Atg7 and Atg8 transcripts), corroborating its importance during metacyclogenesis, endocytosis, by an increase in the expression of the AP-2 complex subunit alpha, protein processing in the endoplasmic reticulum and meiosis. Study findings indicate that in T. cruzi metacyclic trypomastigotes, metabolic processes are decreased, and expression of genes involved in specific cell cycle processes is increased to facilitate transformation to this infective stage.

Los patrones electroforéticos de proteínas salivales permiten diferenciar los grupos transandino y cisandino de las especies de Rhodnius de Colombia / Salivary proteins electrophoretic patterns enabled differentiating Colombian Rhodnius Trans-Andean and Cis-Andean groups

Introducción. Las especies Rhodnius (Hemiptera: Reduviidae: Triatominae) están conformadas por insectos hematófagos vectores de Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, y T. rangeli, parásito infectivo pero no patógeno para el vertebrado. El estudio de la diversidad proteica de la saliva de estos insectos permite la obtención de perfiles electroforéticos unidimensionales característicos de algunas especies de triatominos. Sin embargo, el reporte de los patrones electroforéticos de proteínas salivales de las especies de Rhodnius ha sido escaso. Objetivo. Hacer un análisis comparativo de los perfiles electroforéticos unidimensionales de las proteínas salivales de R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus. Materiales y métodos. Se obtuvieron los perfiles electroforéticos de la saliva de las especies en estudio mediante electroforesis en gel de poliacrilamida con dodecilsulfato sódico (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) y se construyó un fenograma mediante el método UPGMA (Unweighted Pair Group Method Using Arithmetic Averages). Resultados. Los perfiles electroforéticos de las proteínas solubles de saliva presentaron bandas en un rango de masa aproximado de 15 a 45 kDa, los cuales permitieron diferenciar las cinco especies estudiadas. El fenograma reveló la existencia de dos grupos principales: uno conformado por los grupos cisandinos Pictipes y Prolixus y otro constituido por el grupo transandino Pallescens. Conclusiones. Existen diferencias en los perfiles electroforéticos de las proteínas salivales entre R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus, cuya variabilidad permitió construir un fenograma congruente con los grupos del género Rhodnius.

Introduction: Rhodnius (Hemiptera: Reduviidae: Triatominae) species are made up of haematophagous insect vectors for Trypanosoma cruzi (Chagas' disease aetiological agent) and T. rangeli, an infective parasite that is not pathogenic for vertebrate hosts. The study of their salivary protein diversity enables the obtention of characteristic one-dimensional electrophoretic profiles of some triatomine species; however, few reports have dealt with Rhodnius species salivary proteins electrophoretic patterns. Objective: To compare R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus' salivary proteins one-dimensional electrophoretic profiles. Materials and methods: SDS-PAGE was used for obtaining electrophoretic profiles of saliva from the species under study. The unweighted pair group method with arithmetic mean (UPGMA) was used for constructing a phenogram. Results: Electrophoretic profiles of soluble saliva had protein bands ranging from 15 to 45 kDa, thereby enabling the five species studied to be differentiated. The phenogram revealed two main groups, one formed by the Pictipes and Prolixus cis-Andean groups and another consisting of the Pallescens trans-Andean group. Conclusion: Differences were revealed regarding R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus electrophoretic profiles of salivary proteins; their variability facilitated constructing a phenogram which was taxonomically congruent with the groups from the genus Rhodnius.

Slight temperature changes cause rapid transcriptomic responses in Trypanosoma cruzi metacyclic trypomastigotes.

BACKGROUND: Severe changes in temperature can affect the behavior and ecology of some infectious agents. Trypanosoma cruzi is a protozoan that causes Chagas disease. This parasite has high genetic variability and can be divided into six discrete typing units (DTUs). Trypanosoma cruzi also has a complex life-cycle, which includes the process of metacyclogenesis when non-infective epimastigote forms are differentiated into infective metacyclic trypomastigotes (MT). Studies in triatomines have shown that changes in temperature also affect the number and viability of MT. METHODS: The objective of this study was to evaluate how temperature affects the transcriptional profiles of T. cruzi I and II (TcI and TcII) MT by exposing parasites to two temperatures (27 °C and 28 °C) and comparing those to normal culture conditions at 26 °C. Subsequently, RNA-seq was conducted and differentially expressed genes were quantified and associated to metabolic pathways. RESULTS: A statistically significant difference was observed in the number of MT between the temperatures evaluated and the control, TcII DTU was not strongly affected to exposure to high temperatures compared to TcI. Similar results were found when we analyzed gene expression in this DTU, with the greatest number of differentially expressed genes being observed at 28 °C, which could indicate a dysregulation of different signaling pathways under this temperature. Chromosome analysis indicated that chromosome 1 harbored the highest number of changes for both DTUs for all thermal treatments. Finally, gene ontology (GO) analyses showed a decrease in the coding RNAs involved in the regulation of processes related to the metabolism of lipids and carbohydrates, the evasion of oxidative stress, and proteolysis and phosphorylation processes, and a decrease in RNAs coding to ribosomal proteins in TcI and TcII, along with an increase in the expression of surface metalloprotease GP63 in TcII. CONCLUSIONS: Slight temperature shifts lead to increased cell death of metacyclic trypomastigotes because of the deregulation of gene expression of different processes essential for the TcI and TcII DTUs of T. cruzi.

Transcriptomic changes across the life cycle of Trypanosoma cruzi II.

Trypanosoma cruzi is a flagellated protozoan that causes Chagas disease; it presents a complex life cycle comprising four morphological stages: epimastigote (EP), metacyclic trypomastigote (MT), cell-derived trypomastigote (CDT) and amastigote (AM). Previous transcriptomic studies on three stages (EPs, CDTs and AMs) have demonstrated differences in gene expressions among them; however, to the best of our knowledge, no studies have reported on gene expressions in MTs. Therefore, the present study compared differentially expressed genes (DEGs), and signaling pathway reconstruction in EPs, MTs, AMs and CDTs. The results revealed differences in gene expressions in the stages evaluated; these differences were greater between MTs and AMs-PTs. The signaling pathway that presented the highest number of DEGs in all the stages was associated with ribosomes protein profiles, whereas the other related pathways activated were processes related to energy metabolism from glucose, amino acid metabolism, or RNA regulation. However, the role of autophagy in the entire life cycle of T. cruzi and the presence of processes such as meiosis and homologous recombination in MTs (where the expressions of SPO11 and Rad51 plays a role) are crucial. These findings represent an important step towards the full understanding of the molecular basis during the life cycle of T. cruzi.