Phenotypic Variability of the Amazonian Species Rhodnius brethesi (Hemiptera: Reduviidae).

The wild species Rhodnius brethesi (Matta, 1919) (Hemiptera: Reduviidae) is found in areas of piassabais in microregion of Rio Negro, Amazonas, Brazil. Its geographical distribution overlaps the areas of Leopoldinia piassaba palm. In areas where palm trees are found, transmission cycle of Trypanosoma cruzi is related to the extractive activity of the palm fiber, exposing workers to wild vector transmission of this parasite. The close association with the palm tree L. piassaba suggests that this wild triatomine has special features in its sensory system allowing specificity of ecotope. The objective of the study is to identify the antennal sensilla phenotype and morphologically characterize the size and shape of the wings of wild R. brethesi, and to compare with the phenotype present in individuals reared in the laboratory. From the samples taken in the field, the presence of the species R. brethesi was found on both banks of the Rio Negro. The techniques used to verify the morphological patterns are important resources for observations of the triatomine populations, be they in an artificial or natural habitat.

Identification and characterization of microRNAs in the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria (Diptera: Calliphoridae).

MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression through post-transcriptional regulation. Here, we report the identification and characterization of miRNAs in two closely related screwworm flies with different feeding habits: Cochliomyia hominivorax and Cochliomyia macellaria. The New World screwworm, C. hominivorax, is an obligatory parasite of warm-blooded vertebrates, whereas the secondary screwworm, C. macellaria, is a free-living organism that feeds on decaying organic matter. Here, the small RNA transcriptomes of adults and third-instar larvae of both species were sequenced. A total of 110 evolutionarily conserved miRNAs were identified, and 10 putative precursor miRNAs (pre-miRNAs) were predicted. The relative expression of six selected miRNAs was further investigated, including miRNAs that are related to reproduction and neural processes in other insects. Mature miRNAs were also characterized across an evolutionary time scale, suggesting that the majority of them have been conserved since the emergence of the Arthropoda [540 million years ago (Ma)], Hexapoda (488 Ma) and Brachycera (195 Ma) lineages. This study is the first report of miRNAs for screwworm flies. We also performed a comparative analysis with the hereby predicted miRNAs from the sheep blowfly, Lucilia cuprina. The results presented may advance our understanding of parasitic habits within Calliphoridae and assist further functional studies in blowflies.

Ecological diversity of Trypanosoma cruzi transmission in the Amazon basin. The main scenaries in the Brazilian Amazon.

The ecological diversity of Trypanosoma cruzi transmission in the Brazilian Amazon region is directly interlinked with the parasite's extensive reservoir, composed of 33 species of wild mammals within the following orders: Marsupialia, Chiroptera, Rodentia, Xenarthra, Carnivora and Primates; and of 16 species of wild triatomines, of which ten may be infected with T. cruzi. Four scenarios for the diversity of T. cruzi transmission in the Brazilian Amazon region are evident: (i) T. cruzi transmission between vectors and wild mammals, which is characterized as a wild enzooty encompassing the entire Amazon basin; (ii) accidental T. cruzi transmission from vectors and wild mammals to humans, when they invade the wild ecotope or when these vectors and wild mammals invade human homes; (iii) occupational Chagas disease among piassava (Leopoldinia piassaba) palm fiber gatherers, transmitted by the vector Rhodnius brethesi, for which these palm trees are the specific ecotope; (IV) oral T. cruzi transmission to humans through food contamination, particularly in juices from plants such as assai, which today is considered to be endemic in the Brazilian Amazon region, with more than 1500 cases notified.

Molecular phylogenetics of Oestroidea (Diptera: Calyptratae) with emphasis on Calliphoridae: insights into the inter-familial relationships and additional evidence for paraphyly among blowflies.

The superfamily Oestroidea, comprising ∼15,000 species, is a large and ecologically diverse clade within the order Diptera. Among its six commonly recognized families, Calliphoridae seems to be crucial for understanding evolutionary relationships in the group, as it is recognized as a controversial paraphyletic grouping. To further investigate this matter, the ITS2, 28S, COI and 16S regions were used to infer phylogenetic relationships in Oestroidea with maximum-parsimony (MP), maximum-likelihood (ML) and Bayesian inference (BI) methods. For the BI analyses, a deep evaluation of different data partitioning strategies was conducted, including consideration of structural conformation (ITS2 and 16S) and codon position (COI) information. Results suggest the existence of two main clades in Oestroidea: (Tachinidae+Mesembrinellinae) and (Rhiniinae, (Sarcophagidae+Calliphoridae sensu stricto)). Oestridae was recovered as sister group of the remaining Oestroidea in the MP trees while it was placed closer to the (Rhiniinae+Sarcophagidae+Calliphoridae sensu stricto) group in the ML and BI trees. A paraphyletic Calliphoridae was recovered, confirming the exclusion of Rhiniinae, a clade recently promoted to family status and therefore already excluded. Mesembrinellinae could also be considered a distinct group apart from Calliphoridae, although further studies are required. Consideration of structural and codon position information led to a significant increase in the log-likelihoods of the analyses, which were accompanied by small changes in the inferred topologies, branch lengths and posterior probability support values. However, as model complexity increases, so does uncertainty across the estimated parameters, including tree topologies, and phylogenies inferred under very parameter-rich models may be less reliable even when possessing higher log-likelihoods.

Evaluation of the internal transcribed spacer 2 (ITS2) as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies (Diptera: Calliphoridae).

The internal transcribed spacer 2 (ITS2) is a small non-coding region located inside the nuclear ribosomal DNA cluster. ITS2 sequence variability is thought to be appropriate to differentiate species and for phylogenetic reconstructions analyses, which can be further improved if structural information is considered. We evaluated the potential of ITS2 as a molecular marker for phylogenetic inference in Calliphoridae (Diptera: Brachycera) using a broad range of inference methods and different substitution models, accounting or not for structural information. Sequence analyses revealed a hierarchically organized pattern of sequence variation and a small level of nucleotide substitution saturation. Intragenomic variation due to small sequence repeats was found mainly in the most variable domain (IV), but it has no significant impact on the phylogenetic signal at the species level. Inferred secondary structures revealed that GC pairs are more frequently found flanking bulges and loops regions in more conserved domains, thus ensuring structure stability. In the phylogenetic analyses, the use of substitution models accounting for structural information significantly improves phylogenetic inference in both neighbour-joining and Bayesian analyses, although the former provides limited resolution for dealing with highly divergent sequences. For Bayesian analyses, a significant improvement in likelihood was observed when considering structure information, although with small changes in topology and overall support, probably reflecting better evolutionary rates estimates. Based on these findings, ITS2 is a suitable molecular marker for phylogenetic analyses in Calliphoridae, at both species and generic level.

A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA.

We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.

Comparative phylogeography of Trypanosoma rangeli and Rhodnius (Hemiptera: Reduviidae) supports a long coexistence of parasite lineages and their sympatric vectors.

To make reliable interpretations about evolutionary relationships between Trypanosoma rangeli lineages and their insect vectors (triatomine bugs of the genus Rhodnius) and, thus, about the determinant factors of lineage segregation within T. rangeli, we compared phylogenies of parasite isolates and vector species. Sixty-one T. rangeli isolates from invertebrate and vertebrate hosts were initially evaluated in terms of polymorphism of the spliced-leader gene (SL). Further analysis based on SL and SSUrRNA sequences from 33 selected isolates, representative of the overall phylogenetic diversity and geographical range of T. rangeli, supported four phylogenetic lineages within this species. By comparing the phylogeny of Rhodnius species with that inferred for T. rangeli isolates and through analysis of the geographical range of the isolates, we showed that there is a very significant overlap in the distribution of Rhodnius species and T. rangeli lineages. Congruence between phylogeographical analysis of both T. rangeli lineages and complexes of Rhodnius species are consistent with the hypothesis of a long coexistence of parasites and their vectors, with lineage divergence associated with sympatric species of Rhodnius apparently without association with particular vertebrate hosts. Separation of T. rangeli isolates from vectors of distinct complexes living in sympatry favours the absence of gene flow between the lineages and suggests evolution of T. rangeli lineages in independent transmission cycles, probably associated to specific Rhodnius spp. ecotopes. A polymerase chain reaction assay based on SL intergenic sequences was developed for simultaneous identification and lineage genotyping of T. rangeli in epidemiological surveys.

Phylogeny, taxonomy and grouping of Trypanosoma rangeli isolates from man, triatomines and sylvatic mammals from widespread geographical origin based on SSU and ITS ribosomal sequences.

Phylogenetic relationships among Trypanosoma rangeli isolates from man, wild mammals and triatomine bugs from widespread geographical origin were inferred by comparison of the small subunit of ribosomal gene sequences. The phylogenetic trees indicated that the subgenus Herpetosoma is polyphyletic and strongly supported division of this group into two monophyletic lineages, one made up of T. rangeli, T. rangeli-like and allied species and other consisting of T. lewisi and related taxa. Based on phylogenetic analysis, morphology, behaviour in vertebrate and invertebrate hosts and epidemiology we propose: a) the validation of Herpetosoma as a taxon comprised only for species of group lewisi and the maintenance of T. lewisi as the type-species of this subgenus; b) the classification of T. rangeli, T. rangeli-like and allied species into a 'T. rangeli-clade' more closely related to Schizotrypanum than to T. lewisi or T. brucei. The phylogenetic tree disclosed at least 4 groups within the clade T. rangeli, all confirmed by polymorphism of the internal transcribed spacer, thus conferring for the first time phylogenetic support to groups of T. rangeli and corroborating the high complexity of this taxon. Grouping was independent of their mammalian host-species and geographical origin, indicating that other factors are determining this segregation.

Long-term follow-up of co-infected HIV and Trypanosoma cruzi Brazilian patients.

Three cases of Trypanosoma cruzi-HIV co-infected haemophiliacs are described. Parasitological (xenodiagnosis, haemoculture, PCR) and immunological (CD4+ and CD8+ T cell counts, in vitro lymphoproliferative responses) studies were performed. Hybridization of isolated parasites with a specific probe confirmed the T. cruzi aetiology. We observed that despite the high parasitaemia, no clinical or parasitological evidence of T. cruzi reactivation was detected. CD4+ T cells decreased with time in two patients and the lymphocyte proliferative response to T. cruzi was very low in all patients. These data suggest that T. cruzi infection may have a long silent course in immunosuppressed HIV patients. Therefore, this parasitic infection should be investigated in any AIDS patient coming from areas endemic for Chagas' disease.

Randomly amplified polymorphic DNA analysis of Trypanosoma rangeli and allied species from human, monkeys and other sylvatic mammals of the Brazilian Amazon disclosed a new group and a species-specific marker.

We characterized 14 trypanosome isolates from sylvatic mammals (9 from primates, 1 from sloth, 2 from anteaters and 2 from opossum) plus 2 human isolates of Brazilian Amazon. These isolates were proven to be Trypanosoma rangeli by detection of metacyclic trypomastigotes in the salivary glands of triatomines and by a specific PCR assay. Polymorphism determined by randomly amplified polymorphic DNA (RAPD) revealed that most (12) of the Brazilian T. rangeli isolates from the Amazon differed from those of other geographical regions, thus constituting a new group of T. rangeli. Four Brazilian isolates clustered together with a previously described group (A) that was described as being composed of isolates from Colombia and Venezuela. Isolates from Panama and El Salvador form another group. The isolate from Southern Brazil did not cluster to any of the above-mentioned groups. This is the first study that assesses the genetic relationship of a large number of isolates from wild mammals, especially from non-human primates. A randomly-amplified DNA fragment (Tra625) exclusive to T. rangeli was used to develop a PCR assay able to detect all T. rangeli groups.

Methods for the recovery of mitochondrial DNA sequences from museum specimens of myiasis-causing flies.

Mitochondrial DNA (mtDNA) sequences from eight species of myiasis-causing flies, stored for up to 50 years, were amplified successfully. Universal primers were used to amplify six specific regions from total genomic DNA, including five mtDNA genes. The comparison of phenol/chloroform, DNAzol and Chelex techniques for DNA extraction showed that the DNAzol reagent was the most efficient in retrieving DNA from museum specimens, although the Chelex extraction procedure is currently the most frequently reported method. Comparison of the universal primer sequences with the homologous sequences of Cochliomyia hominivorax Coquerel and Chrysomya putoria Wiedemann (Diptera: Calliphoridae) revealed mismatches that could contribute to the low recovery of a short sequence from subunit II of cytochrome oxidase. The ability to characterize mtDNA markers from museum specimens should be useful in comparative studies of contemporary samples and should help in elucidating species introduction, colonization and dispersal.

Fungal flora of the digestive tract of 5 species of triatomines vectors of Trypanosoma cruzi, Chagas 1909.

A study of the mycobiota in the digestive tract of 5 important species of triatomines, Triatoma brasiliensis, T infestans, T. sordida, T. pseudomaculata and T. vitticeps, was made. The digestive tracts of 164 adults and 535 nymphs of those triatomines were studied and 393 fungal strains were isolated. The genera with the greatest number of species were Penicillium (19 species), Aspergillus (17 species) and Acremonium (5 species) and the most frequent species, in decreasing order, were Penicillium corylophilum, Aspergillus niger, Penicillium felluttanum, Cladosporium herbarum, Penicillium waksmanii, Aspergillus awamori and Paecilomyces variotii. Among the isolated fungi, we found species that are recognized as entomopathogenic and pathogenic for humans and animals.

Fungal flora of the digestive tract of Panstrongylus megistus (Reduviidae) used for experimental xenodiagnosis of Trypanosoma (Schizotripanum) cruzi Chagas, 1909.

A survey of the fungi isolated from the digestive tract of Panstrongylus megistus (insects vectors from Chagas' disease) used on xenodiagnosis was carried out. Two hundred and fourteen fungal strains were isolated from 180 nymphs. Aspergillus and Penicillium were the most predominant genera and some of their species were new records concerning insects. A great reduction in the fungal population was observed in the material that was positive for Trypanosoma cruzi.

The mitochondrial genome of the primary screwworm fly Cochliomyia hominivorax (Diptera: Calliphoridae).

The complete sequence of the mitochondrial genome of the screwworm Cochliomyia hominivorax was determined. This genome is 16,022 bp in size and corresponds to a typical Brachycera mtDNA. A Serine start codon for COI and incomplete termination codons for COII, NADH 5 and NADH 4 genes were described. The nucleotide composition of C. hominivorax mtDNA is 77% AT-rich, reflected in the predominance of AT-rich codons in protein-coding genes. Non-optimal codon usage was commonly observed in C. hominivorax mitochondrial genes. Phylogenetic analysis distributed the Acalypterate species as a monophyletic group and assembled the C. hominivorax (Calyptratae) and the Acalyptratae in a typical Brachycera cluster. The identification of diagnostic restriction sites on the sequenced mitochondrial genome and the correlation with previous RFLP analysis are discussed.

[Reactivation of Trypanosoma cruzi infection in patients with acquired immunodeficiency syndrome]. / Reativação da infecção por Trypanosoma cruzi em paciente com síndrome de imunodeficiência adquirida.

A patient with AIDS and asymptomatic Chagas's disease and positive xenodiagnosis was taking ketoconazole in order to suppress parasitemia and prevent reactivation of Chagas's disease. Ketoconazole was unplanned suspended after 6 months, and the patient was admitted with fever, headache, vomiting, tachycardia, postural hypotension, hepatosplenomegaly, and positive xenodiagnosis one month later. Treatment with benzonidazole was begun leading to suppression of parasitemia. The patient had probability a neurotoxoplasmosis associated and progressed to coma and death with sepsis. No parasite was found in autopsy.

Chagas disease: from bush to huts and houses. Is it the case of the Brazilian Amazon?

Two of the major problems facing the Amazon - human migration from the other areas and uncontrolled deforestation - constitute the greatest risk for the establishment of endemic Chagas disease in this part of Brazil. At least 18 species of triatomines had been found in the Brazilian Amazon, 10 of them infected with Trypanosoma cruzi, associated with numerous wild reservoirs. With wide-range deforestation, wild animals will perforce be driven into other areas, with tendency for triatomines to become adapted to alternative food sources in peri and intradomicilies. Serological surveys and cross-sectional studies for Chagas disease, carried out in rural areas of the Rio Negro, in the Brazilian Amazon, showed a high level of seropositivity for T. cruzi antibodies. A strong correlation of seroreactivity with the contact of gatherers of piaçava fibers with wild triatomines could be evidenced.

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences.

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24S alpha ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24S alpha rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage 1 was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite.