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.

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.

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.

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.

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.

Comparison of the polymerase chain reaction with two classical parasitological methods for the diagnosis of Chagas disease in an endemic region of north-eastern Brazil.

The sensitivities for Chagas disease diagnosis of haemoculture, xenodiagnosis, and polymerase chain reaction (PCR) amplification of Trypanosoma cruzi kinetoplast deoxyribonucleic acid (DNA) were compared for 101 patients living in an endemic region who were serologically positive for T. cruzi. PCR gave 60 positive results (59.4%), while a haemoculture was positive in 26 cases (25.7%) and xenodiagnosis in 36 (35.6%). Four xenodiagnosis-positive but PCR-negative patients were examined in detail. The discrepancies were not due to inhibition of the PCR reactions, as the samples were used successfully to amplify a human sequence. Nor were they due to a variation in kinetoplast DNA sequences, as the kinetoplast DNA of the parasite strains isolated from these patients after xenodiagnosis gave rise to the expected product when amplified by the PCR. We concluded that no parasite was present in the 5 mL of blood used for PCR, while probably a single T. cruzi cell was present in the blood volume ingested by the insects during xenodiagnosis (about 3 mL). This suggests that the total blood quantity collected for the PCR may be important with patients with low parasitaemia.

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.

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.

Chagas' disease in the Brazilian Amazon. I--A short review.

At least eighteen species of triatominae have been found in the Brazilian Amazon, nine of them naturally infected with Trypanosoma cruzi or "cruzi-like" trypanosomes and associated with numerous wild reservoirs. Despite the small number of human cases of Chagas' disease described to date in the Brazilian Amazon the risk that the disease will become endemic in this area is increasing for the following reasons: a) uncontrolled deforestation and colonization altering the ecological balance between reservoir hosts and wild vectors; b) the adaptation of reservoir hosts of T. cruzi and wild vectors to peripheral and intradomiciliary areas, as the sole feeding alternative; c) migration of infected human population from endemic areas, accompanied by domestic reservoir hosts (dogs and cats) or accidentally carrying in their baggage vectors already adapted to the domestic habitat. In short, risks that Chagas' disease will become endemic to the Amazon appear to be linked to the transposition of the wild cycle to the domestic cycle in that area or to transfer of the domestic cycle from endemic areas to the Amazon.

[Xenodiagnosis in chronic Chagas' disease. I. The sensitivity of Panstrongylus megistus and Triatoma infestans]. / Xenodiagnóstico na doença de Chagas crônica. I--Sensibilidade de Panstrongylus megistus e Triatoma infestans.

From January 1986 to February 1994, 563 xenodiagnosis (XD) were applied in 563 chronic chagasic patients from different areas of Brazil; 292 were women and 271 were men between 6 and 89 years (average: 41.4 +/- 14.7 years). To each XD 40 nymphs on the 4th stage were used: 20 from Panstrongylus megistus (Pm) and 20 from Triatoma infestans (Ti) in fast, during at least 14 days. The exam in each nymph was made 45 days after being applied on the patient, by observation in optical microscopy of the drugs and/or the grinded from the digestive tube. The results are: a) 205 (36.4%) positive XD, including 85 (15.1%) due only nymphs of Pm, 44 (7.8%) Ti and 76 (13.5%) Pm and Ti; b) positively in 4.9% of the nymphs from Pm and in 3.0% of the Ti nymphs examined. These results' analysis showed that the Pm nymphs were more sensitive that Ti's ones to the infection by Trypanosoma cruzi, increasing considerably the xenopositivity, independently from birthplace, sex or age of the patients. These results point out that to increase the efficacy of XD in chronic Chagas' disease, the exam must have more than a species of triatomine with different sensibilities to the T. cruzi infection, and in case of using one species on XD, Pm must substitute Ti.

[Morbidity of Chagas disease in areas of Sertão da Paraiba and Caatinga do Piauí]. / Morbidade da doença de Chagas em áreas do Sertão da Paraíba e da Caatinga do Piauí.

A clinical and electrocardiographic case control study was carried out with 186 pairs of persons with positive and negative serology for T. cruzi infection from the Sertão Paraíba and in 200 seropositive cases from the region of Caatinga in the State of Piauí, North-eastern Brazil. The predominant clinical manifestations in seropositive cases in both areas were: palpitations, dyspnea on effort, precordial pain, dysphagia, odynophagia, pyrosis and intestinal constipation. The EKG abnormalities rates suggestive of chronic chagasic cardiopathy were respectively in Paraíba and Piauí: AV block 3.8% and 2%, RBBB III 6.4% and 7%, RBBB III+ LAB 10.7% and 10.5%, and multifocal extrasystoles 2.7% and 3%. Xenodiagnosis in a sample of 54 seropositive individuals in the Sert-ao of Para-iba and in 120 in the Caatinga of Piauí was revealed 13% and 34% positive; PCR tests in a sample of 47 seropositives in Paraíba and 101 in Piauí revealed positives in 44.6% and 59.5% respectively. Blood culture in LIT media of 101 seropositive cases from the Caatinga of Piauí was positive for T. cruzi in 25.7%. A triatomine survey carried out in a sample of 132 domiciles and peridomiciles in the Sertão of Paraíba and in a sample of 159 in the Caatinga of Piauí showed the following results: In Paraíba, 16 specimens of T. brasiliensis, not infected with T. cruzi, were captured. In Piauí, 750 triatomines were captured, of these 625 were examined: 49 were T. pseudomaculata, not infected with T. cruzi (19 in peridomiciles and 30 in the domiciles), and 576 were T. brasiliensis (371 in the domiciles and 205 in the peridomiciles) and of this latter specie 32 (5.5%) were infected with T. cruzi (31 in the domiciles and one in the peridomicile).

[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.

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.

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.

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.

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.

Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease.

Immunoblotting with trypomastigote excreted-secreted antigens (TESA blot) of Trypanosoma cruzi was evaluated as a method for diagnosis of chronic and acute phases as well as congenital (in newborn children) Chagas' disease. Serum samples from acute-phase and congenital infections were considered to be positive when they reacted with ladder-like bands of 130- to 200-kDa antigens, recognized by immunoglobulin M (IgM) and IgG antibodies, while IgG from chronic-phase sera recognized a broad band antigen of 150 to 160 kDa. Nonchagasic sera were not reactive to these antigens. The study was carried out on 512 patients, 111 of whom were nonchagasic but included cases of leishmaniasis or other pathologies, and 401 chagasic patients. The latter group comprised 361 chronic cases, 36 acute cases, and 4 congenital cases in newborn children. Among the chronic cases, 256 were from areas in which T. cruzi is endemic but which differed widely in the pathogenic expression of T. cruzi infection and in parasitemia levels. These patients at the same time showed a broad range of low, medium, and high reactivity to conventional enzyme-linked immunosorbent assays and indirect immunofluorescence serotests for Chagas' disease. For these reasons they may better represent the universe of chagasic patients than would a sample of highly reactive sera obtained from chagasic patients in a single area endemic for T. cruzi. All acute and congenital cases showed positivity in the IgM and IgG TESA blots, while chronic cases were 100% positive for IgG antibodies. In nonchagasic sera, including 30 cases of visceral and muco-cutaneous leishmaniasis, the specificity index was 1.000, and no cross-reactions were observed. The TESA blot thus seems to be useful as a sensitive and specific diagnostic assay in cases of suspected acute or congenital T. cruzi infection and as a general confirmatory test for conventional Chagas' disease serology.