Microevolution of Trypanosoma cruzi reveals hybridization and clonal mechanisms driving rapid genome diversification.

Protozoa and fungi are known to have extraordinarily diverse mechanisms of genetic exchange. However, the presence and epidemiological relevance of genetic exchange in Trypanosoma cruzi, the agent of Chagas disease, has been controversial and debated for many years. Field studies have identified both predominantly clonal and sexually recombining natural populations. Two of six natural T. cruzi lineages (TcV and TcVI) show hybrid mosaicism, using analysis of single-gene locus markers. The formation of hybrid strains in vitro has been achieved and this provides a framework to study the mechanisms and adaptive significance of genetic exchange. Using whole genome sequencing of a set of experimental hybrids strains, we have confirmed that hybrid formation initially results in tetraploid parasites. The hybrid progeny showed novel mutations that were not attributable to either (diploid) parent showing an increase in amino acid changes. In long-term culture, up to 800 generations, there was a variable but gradual erosion of progeny genomes towards triploidy, yet retention of elevated copy number was observed at several core housekeeping loci. Our findings indicate hybrid formation by fusion of diploid T. cruzi, followed by sporadic genome erosion, but with substantial potential for adaptive evolution, as has been described as a genetic feature of other organisms, such as some fungi.

Repeat-Driven Generation of Antigenic Diversity in a Major Human Pathogen, Trypanosoma cruzi.

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan parasite, is the causative agent of American trypanosomiasis (Chagas disease). Having a very plastic, repetitive and complex genome, the parasite displays a highly diverse repertoire of surface molecules, with pivotal roles in cell invasion, immune evasion and pathogenesis. Before 2016, the complexity of the genomic regions containing these genes impaired the assembly of a genome at chromosomal level, making it impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here describe the genome assembly of the Sylvio X10/1 genome sequence, which since 2016 has been used as a reference genome sequence for T. cruzi clade I (TcI), produced using high coverage PacBio single-molecule sequencing. It was used to analyze deep Illumina sequence data from 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene distribution showed the unusual duality in the organization of the parasite genome, a synteny of the core genomic region with related protozoa flanked by unique and highly plastic multigene family clusters encoding surface antigens. The presence of abundant interspersed retrotransposons in these multigene family clusters suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response on these TcI strains. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure.

IncX4 Plasmid-Mediated mcr-1.1 in Polymyxin-Resistant Escherichia coli from Outpatients in Santa Catarina, Southern Brazil.

Plasmid-mediated polymyxin resistance has become a global health concern, not only because its dissemination has occurred drastically but also because it has begun to be reported in multidrug-resistant (MDR) pathogens. We hereby report microbiological and genomic characteristics of two mcr-1.1-positive polymyxin-resistant Escherichia coli isolates identified for the first time in community patients, in Santa Catarina, Southern Brazil. E. coli strains belonging to ST206 and ST354 and the resistome analysis revealed the presence of clinically important genes responsible for MDR profile. Interestingly, in both polymyxin-resistant E. coli strains, mcr-1.1 genes were carried by IncX4 plasmids, responsible for the worldwide dissemination of mcr-type genes. In this regard, plasmid backbones were almost identical to the first IncX4 plasmid reported in Brazil and sharing more than 99.9% identity to IncX4 plasmids from China, also lacking the ISApl1 insertion sequence upstream of mcr-1. In conclusion, these data confirm the presence of international ST206 and ST354 carrying mcr-1.1 genes and that the IncX4 plasmids have been key vectors contributing to the endemic status of mcr-1.1-positive polymyxin-resistant E. coli in Brazil. Also, we described the first known clinical isolate with the mrc1.1 gene in Santa Catarina state, Brazil, showing that plasmid-mediated polymyxin resistance has been affecting humans earlier than has been known so far.

Isoniazid-induced control of Mycobacterium tuberculosis by primary human cells requires interleukin-1 receptor and tumor necrosis factor.

Proinflammatory cytokines are critical mediators that control Mycobacterium tuberculosis (Mtb) growth during active tuberculosis (ATB). To further inhibit bacterial proliferation in diseased individuals, drug inhibitors of cell wall synthesis such as isoniazid (INH) are employed. However, whether INH presents an indirect effect on bacterial growth by regulating host cytokines during ATB is not well known. To examine this hypothesis, we used an in vitro human granuloma system generated with primary leukocytes from healthy donors adapted to model ATB. Intense Mtb proliferation in cell cultures was associated with monocyte/macrophage activation and secretion of IL-1ß and TNF. Treatment with INH significantly reduced Mtb survival, but altered neither T-cell-mediated Mtb killing, nor production of IL-1ß and TNF. However, blockade of both IL-1R1 and TNF signaling rescued INH-induced killing, suggesting synergistic roles of these cytokines in mediating control of Mtb proliferation. Additionally, mycobacterial killing by INH was highly dependent upon drug activation by the pathogen catalase-peroxidase KatG and involved a host PI3K-dependent pathway. Finally, experiments using coinfected (KatG-mutated and H37Rv strains) cells suggested that active INH does not directly enhance host-mediated killing of Mtb. Our results thus indicate that Mtb-stimulated host IL-1 and TNF have potential roles in TB chemotherapy.

Genotyping of Trypanosoma cruzi DTUs and Trypanosoma rangeli genetic groups in experimentally infected Rhodnius prolixus by PCR-RFLP.

The specific detection and genetic typing of trypanosomes that infect humans, mammalian reservoirs, and vectors is crucial for diagnosis and epidemiology. We utilized a PCR-RFLP assay that targeted subunit II of cytochrome oxidase and 24Sα-rDNA to simultaneously detect and discriminate six Trypanosoma cruzi discrete typing units (DTUs) and two genetic groups of Trypanosoma rangeli (KP1+/KP1-) in intestinal contents of experimentally infected Rhodnius prolixus. The PCR assays showed that in 23 of 29 (79.4%) mixed infections with the six T. cruzi DTUs and mixed infections with individual DTUs and/or groups KP1+ and KP1-, both parasites were successfully detected. In six mixed infections that involved TcIII, the TcI, TcII, TcV, and TcVI DTUs predominated to the detriment of TcIII, indicating the selection of genetic groups. Interactions between different genetic groups and vectors may lead to genetic selection over TcIII. The elimination of this DTU by the immune system of the vector appears unlikely because TcIII was present in other mixed infections (TcIII/TcIV and TcIII/KP1+). Both molecular markers used in this study were sensitive and specific, demonstrating their usefulness in a wide geographical area where distinct genotypes of these two species are sympatric. Although the cellular and molecular mechanisms that are involved in parasite-vector interactions are still poorly understood, our results indicate a dynamic selection toward specific T. cruzi DTUs in R. prolixus during mixed genotype infections.

The Comparative Genomics and Phylogenomics of Leishmania amazonensis Parasite.

Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the "Mexicana complex", reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development.

STINGRAY: system for integrated genomic resources and analysis.

BACKGROUND: The STINGRAY system has been conceived to ease the tasks of integrating, analyzing, annotating and presenting genomic and expression data from Sanger and Next Generation Sequencing (NGS) platforms. FINDINGS: STINGRAY includes: (a) a complete and integrated workflow (more than 20 bioinformatics tools) ranging from functional annotation to phylogeny; (b) a MySQL database schema, suitable for data integration and user access control; and (c) a user-friendly graphical web-based interface that makes the system intuitive, facilitating the tasks of data analysis and annotation. CONCLUSION: STINGRAY showed to be an easy to use and complete system for analyzing sequencing data. While both Sanger and NGS platforms are supported, the system could be faster using Sanger data, since the large NGS datasets could potentially slow down the MySQL database usage. STINGRAY is available at http://stingray.biowebdb.org and the open source code at http://sourceforge.net/projects/stingray-biowebdb/.

Trans-sialidase stimulates eat me response from epithelial cells.

Epithelial cell invasion by the protozoan parasite Trypanosoma cruzi is enhanced by the presence of an enzyme expressed on its cell surface during the trypomastigote life cycle stage. The enzyme, trans-sialidase (TS), is a member of one of the largest gene families expressed by the parasite and the role of its activity in mediating epithelial cell entry has not hitherto been understood. Here we show that the T. cruzi TS generates an eat me signal which is capable of enabling epithelial cell entry. We have utilized purified, recombinant, active (TcTS) and inactive (TcTS2V0) TS coated onto beads to challenge an epithelial cell line. We find that TS activity acts upon G protein coupled receptors present at the epithelial cell synapse with the coated bead, thereby enhancing cell entry. By so doing, we provide evidence that TS proteins bind glycans, mediate the formation of distinct synaptic domains and promote macropinocytotic uptake of microparticles into a perinuclear compartment in a manner which may emulate entosis.

Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family.

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Outbreak of autochthonous canine visceral leishmaniasis in Santa Catarina, Brazil / Surto autóctone de leishmaniose visceral canina no Estado de Santa Catarina

The present study reports the first outbreak of autochthonous canine visceral leishmaniasis in Florianópolis, Santa Catarina, southern Brazil. Following the report of two cases of CVL, the Control Center of Zoonotic Diseases conducted a serological survey by ELISA and IFAT assays in seven districts of the Santa Catarina Island. Eleven seropositive dogs of autochthonous transmission were used in the present study. Infection by Leishmania sp. was confirmed by parasitological examination of bone marrow, liver, spleen and lymph nodes, culture in Schneider's medium and PCR. Leishmania sp. isolates were characterized by PCR-RFLP and hybridization with specific probes, allowing for the identification of Leishmania infantum. Autochthonous transmission of this disease in an area with high tourist traffic presents a major public health concern and signifies the emergence of an important zoonosis in southern Brazil. Therefore, the implementation of surveillance and control measures is imperative to prevent the spread of the disease among the canine population as well as transmission to the human population.

O presente estudo relata o primeiro surto autóctone de leishmaniose visceral canina (LCV) em Florianópolis, Santa Catarina, Brasil. Durante levantamento soro-epidemiológico realizado pelo Centro de Controle de Doenças Zoonóticas (CCZ) envolvendo 2.124 cães, 29 (1,37%) foram soropositivos para VL (ELISA + RIFI). Onze cães positivos por transmissão autóctone foram utilizados no presente estudo. A confirmação da infecção por Leishmania sp. foi realizada pelo exame parasitológico da medula óssea, fígado, baço e linfonodos, cultura em meio Schneider e PCR. Os isolados de Leishmania sp. foram caracterizados por PCR-RFLP e hibridação com sondas específicas, permitindo a identificação de Leishmania infantum. A transmissão autóctone da LCV em uma área com grande fluxo turístico como Florianópolis representa um preocupante risco à saúde pública e o surgimento de uma importante zoonose no sul do Brasil. Neste contexto, a implementação de medidas de vigilância e controle da doença são fundamentais para evitar a propagação da doença entre a população canina, bem como a transmissão para a população humana.

Sequence analysis of the spliced-leader intergenic region (SL-IR) and random amplified polymorphic DNA (RAPD) of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis, R. colombiensis, R. pallescens and R. prolixus suggests a degree of co-evolution between parasites and vectors.

Spliced leader intergenic region (SL-IR) sequences from 23 Trypanosoma rangeli strains isolated from the salivary glands of Rhodnius colombiensis, R. ecuadoriensis, R. pallescens and R. prolixus and two human strains revealed the existence of 4 genotypes with CA, GT, TA, ATT and GTAT microsatellite repeats and the presence of insertions/deletions (INDEL) and single nucleotide polymorphism (SNP) characterizing each genotype. The strains isolated from the same vector species or the same Rhodnius evolutionary line presented the same genotypes, even in cases where strains had been isolated from vectors captured in geographically distant regions. The dendrogram constructed from the SL-IR sequences separated all of them into two main groups, one with the genotypes isolated from R. prolixus and the other group containing three well defined sub-groups with the genotypes isolated from R. pallescens, R. colombiensis and R. ecuadoriensis. Random amplified polymorphic DNA (RAPD) analysis showed the same two main groups and sub-groups supporting strict T. rangeli genotypes' association with Rhodnius species. Combined with other studies, these results suggest a possible co-evolutionary association between T. rangeli genotypes and their vectors.

HIV-1 genetic diversity and drug resistance among treatment naïve patients from Southern Brazil: an association of HIV-1 subtypes with exposure categories.

BACKGROUND: The AIDS epidemic in Southern Brazil has unique features, showing co-circulation of HIV-1 subtypes C, B and recombinant forms. Florianópolis has the second highest AIDS incidence among Brazilian capitals, but limited information is available about HIV molecular epidemiology and prevalence of primary drug resistance. OBJECTIVES: To investigate the molecular epidemiology of HIV-1 in Florianópolis and to describe the prevalence of primary HIV-1 drug resistance mutations (DMRs). STUDY DESIGN: Epidemiological and clinical data from 82 untreated patients from Florianópolis (2008-2009) were analyzed. The HIV-1 subtype at envelope, protease, reverse transcriptase and integrase regions were determined by phylogenetic and bootscaning analyses and the drug resistance profile were analyzed at the Stanford HIV Drug Resistance Database. RESULTS: The most frequent HIV-1 genetic form was subtype C (65.8%) followed by mosaics BC (18.3%), subtype B (13.4%), subtype F1 (1.2%) and BCF1 recombinant (1.2%). HIV-1 subtype C and BC recombinants were much more frequent in the heterosexual exposure category, whereas subtype B was more common in the MSM exposure category. DRMs were seen in 11% of the sequences, 2.4% of them were related to PI, 5% to NRTI, 3.6% to NNRTI and 1.2% was related to INTI. CONCLUSIONS: The present study confirms the high prevalence of subtype C and BC recombinants in Santa Catarina State and revealed a significant difference in the subtype distribution among distinct virus exposure categories. This study also shows a relative high prevalence of protease/reverse transcriptase primary drug resistance mutations and corroborates the usefulness of the integrase inhibitors in southern Brazil.

Genome of Herbaspirillum seropedicae strain SmR1, a specialized diazotrophic endophyte of tropical grasses.

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme--GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.

Detection of infectious myonecrosis virus in penaeid shrimps using immunoassays: usefulness of monoclonal antibodies directed to the viral major capsid protein.

Despite the economic impact of the infectious myonecrosis virus (IMNV) on shrimp farms in several countries, no method for immunological detection is currently available. With the aim of developing immunodiagnostic methods for IMNV detection in infected shrimps, a recombinant fragment of the IMNV major capsid protein gene encoding amino acids 105-297 (rIMNV105₋297 was heterologously expressed in Escherichia coli and used to immunize Balb/c mice, generating monoclonal antibodies (MAbs). Six hybridomas were obtained, and four of these recognized the presence of IMNV in tissue homogenates from naturally infected shrimps by immunodot blot assay. Among these MAbs, three were able to detect a ~100-kDa protein, which corresponds to the predicted mass of the IMNV major capsid protein, as well as viral inclusion bodies in muscle fibroses by western blot and immunohistochemistry. Two MAbs showed high specificity and sensitivity, showing no cross-reaction with healthy shrimp tissues in any assays, indicating their usefulness for IMNV detection.

Detection of major capsid protein of infectious myonecrosis virus in shrimps using monoclonal antibodies.

Infectious myonecrosis virus (IMNV) has been causing a progressive disease in farm-reared shrimps in Brazil and Indonesia. Immunodiagnostic methods for IMNV detection, although reliable, are not employed currently because monoclonal antibodies (MAbs) against this virus are not available. In this study, a fragment of the IMNV major capsid protein gene, comprising amino acids 300-527 (IMNV(300-527)), was cloned and expressed in Escherichia coli. The nucleotide sequence of the recombinant IMNV(300-527) fragment displayed a high degree of identity to the major capsid protein of IMNV isolates from Brazil (99%) and Indonesia (98%). Ten MAbs were generated against the expressed fragment, and eight of these, mostly IgG(2a) or IgG(2b), were able to bind to IMNV in tissue extracts from shrimps infected naturally in immunodot-blot assays. Six of these MAbs recognized a approximately 100 kDa protein in a Western-blot, which is the predicted mass of IMNV major capsid protein, and also bound to viral inclusions present in muscle fibroses and in coagulative myonecrosis, as demonstrated by immunohistochemistry. Among all those MAbs created, four did not cross-react with non-infected shrimp tissues; this observation supports their applicability as a sensitive and specific immunodiagnosis of IMNV infection in shrimps.

Transcriptomic analyses of the avirulent protozoan parasite Trypanosoma rangeli.

Two species of the genus Trypanosoma infective to humans have been extensively studied at a cell and molecular level, but study of the third, Trypanosoma rangeli, remains in relative infancy. T. rangeli is non-pathogenic, but is frequently mistaken for the related Chagas disease agent Trypanosoma cruzi with which it shares vectors, hosts, significant antigenicity and a sympatric distribution over a wide geographical area. In this study, we present the T. rangeli gene expression profile as determined by the generation of ESTs (Expressed Sequence Tags) and ORESTES (Open Reading Frame ESTs). A total of 4208 unique high quality sequences were analyzed, composed from epimastigote and trypomastigote forms of SC-58 and Choachí strains, representing the two major phylogenetic lineages of this species. Comparative analyses with T. cruzi and other parasitic kinetoplastid species allowed the assignment of putative biological functions to most of the sequences generated and the establishment of an annotated T. rangeli gene expression database. Even though T. rangeli is apathogenic to mammals, genes associated with virulence in other pathogenic kinetoplastids were found. Transposable elements and genes associated mitochondrial gene expression, specifically RNA editing components, are also described for the first time. Our studies confirm the close phylogenetic relationship between T. cruzi and T. rangeli and enable us to make an estimate for the size of the T. rangeli genome repertoire ( approximately 8500 genes).

Differential gene expression in Poecilia vivipara exposed to diesel oil water accommodated fraction.

Diesel fuel is a potential contaminant of estuarine and mangrove areas, particularly because it is the main fuel used in small boats and larger vessels. The aim of this work was to identify genes differentially expressed in the liver of Poecilia vivipara (Guppy) exposed to 10% diesel fuel water accommodated fraction (WAF), employing the subtractive suppressive hybridization (SSH) method. The results showed 27 differentially expressed gene fragments, 12 up-regulated and 15 down-regulated. Among the up-regulated genes were CYP1A, UDPGT1a, ABCC4, Methyltransferase and Apolipoprotein A1. Down-regulated genes included Vitellogenins, C1 Inhibitor and Complement Component 3c. The identified genes are associated with different metabolic functions like biotransformation, membrane transport and immune system, indicating the susceptibility and/or molecular responses of this organism to the toxic effects elicited by diesel fuel WSF.

Transcriptome analysis of Taenia solium cysticerci using Open Reading Frame ESTs (ORESTES).

BACKGROUND: Human infection by the pork tapeworm Taenia solium affects more than 50 million people worldwide, particularly in underdeveloped and developing countries. Cysticercosis which arises from larval encystation can be life threatening and difficult to treat. Here, we investigate for the first time the transcriptome of the clinically relevant cysticerci larval form. RESULTS: Using Expressed Sequence Tags (ESTs) produced by the ORESTES method, a total of 1,520 high quality ESTs were generated from 20 ORESTES cDNA mini-libraries and its analysis revealed fragments of genes with promising applications including 51 ESTs matching antigens previously described in other species, as well as 113 sequences representing proteins with potential extracellular localization, with obvious applications for immune-diagnosis or vaccine development. CONCLUSION: The set of sequences described here will contribute to deciphering the expression profile of this important parasite and will be informative for the genome assembly and annotation, as well as for studies of intra- and inter-specific sequence variability. Genes of interest for developing new diagnostic and therapeutic tools are described and discussed.

Assessing the molecular divergence between Anopheles (Kerteszia) cruzii populations from Brazil using the timeless gene: further evidence of a species complex.

BACKGROUND: Anopheles (Kerteszia) cruzii was the most important vector of human malaria in southern Brazil between 1930-1960. Nowadays it is still considered an important Plasmodium spp. vector in southern and south-eastern Brazil, incriminated for oligosymptomatic malaria. Previous studies based on the analysis of X chromosome banding patterns and inversion frequencies in An. cruzii populations from these areas have suggested the occurrence of three sibling species. In contrast, two genetically distinct groups among An. cruzii populations from south/south-east and north-east Brazil have been revealed by isoenzyme analysis. Therefore, An. cruzii remains unclear. METHODS: In this study, a partial sequence of the timeless gene (approximately 400 bp), a locus involved in the control of circadian rhythms, was used as a molecular marker to assess the genetic differentiation between An. cruzii populations from six geographically distinct areas of Brazil. RESULTS: The timeless gene revealed that An. cruzii from Itaparica Island, Bahia State (north-east Brazil), constitutes a highly differentiated group compared with the other five populations from south and south-east Brazil. In addition, significant genetic differences were also observed among some of the latter populations. CONCLUSION: Analysis of the genetic differentiation in the timeless gene among An. cruzii populations from different areas of Brazil indicated that this malaria vector is a complex of at least two cryptic species. The data also suggest that further work might support the occurrence of other siblings within this complex in Brazil.

Comparative analysis of Trypanosoma rangeli histone H2A gene intergenic region with distinct intraspecific lineage markers.

This study shows the characterization of the histone H2A intergenic region sequences (H2A IR) from Trypanosoma rangeli KP1(+) and KP1(-) strains isolated from distinct hosts and geographic regions. Also, a comparative unweighted pair-group method using arithmetic averages (UPMGA) analysis with polymerase chain reaction profiles of the 24Salpha rDNA and the miniexon genes was performed. Detailed H2A IR sequence analysis revealed a discrete size polymorphism among T. rangeli strains and the presence of single-nucleotide polymorphisms and minisatellite repeats, exclusively allowing an interspecific differentiation from T. cruzi strains representing the main parasite lineages. Differently from the H2A IR, UPMGA analysis of the 24Salpha rDNA and the miniexon genes profiles clearly branched T. rangeli strains into KP1(-) and KP1(+) lineages, clustering separately the Brazilian and Colombian KP1(-) strains. The evolutionary implications of these findings are discussed.