Spatial Distribution of Heterochromatin Bodies in the Nuclei of Triatoma infestans (Klug).

Constitutive heterochromatin typically exhibits low gene density and is commonly found adjacent or close to the nuclear periphery, in contrast to transcriptionally active genes concentrated in the innermost nuclear region. In Triatoma infestans cells, conspicuous constitutive heterochromatin forms deeply stained structures named chromocenters. However, to the best of our knowledge, no information exists regarding whether these chromocenters acquire a precise topology in the cell nuclei or whether their 18S rDNA, which is important for ribosome function, faces the nuclear center preferentially. In this work, the spatial distribution of fluorescent Feulgen-stained chromocenters and the distribution of their 18S rDNA was analyzed in Malpighian tubule cells of T. infestans using confocal microscopy. The chromocenters were shown to be spatially positioned relatively close to the nuclear periphery, though not adjacent to it. The variable distance between the chromocenters and the nuclear periphery suggests mobility of these bodies within the cell nuclei. The distribution of 18S rDNA at the edge of the chromocenters was not found to face the nuclear interior exclusively. Because the genome regions containing 18S rDNA in the chromocenters also face the nuclear periphery, the proximity of the chromocenters to this nuclear region is not assumed to be associated with overall gene silencing.

Histone acetylation and methylation marks in chromatin of Panstrongylus megistus (Hemiptera, Reduviidae).

Panstrongylus megistus, a potential vector of Chagas disease, currently occupies a wider geographic distribution in Brazil than Triatoma infestans, another member of the hemipteran Reduviidae family and a vector of the same disease. A small heterochromatic body (chromocenter) formed by the Y chromosome is evident in the somatic cells of P. megistus, differing in size and chromosome type contribution from the well-studied chromocenters present in T. infestans. While the overall distribution of histone epigenetic marks differ when comparing the heterochromatin and euchromatin territories in T. infestans, no similar data have been established for other hemipteran reduviids, including P. megistus. In the present work, histone acetylation and methylation marks were investigated in cells of Malpighian tubules of P. megistus 5th instar nymphs using immunocytochemical assays and compared to previously published data for T. infestans. Although similarities between these species were found regarding absence of acetylated H3K9, H4K8 and H4K16, and H3K9me and H3K9me2 in the chromocenter, presence of these marks in euchromatin, and presence of H3K9me3 in the chromocenter, no intimate association of acetylated H4K8 and 18S rDNA was revealed in the chromocenter of P. megistus. The elevated abundance of H3K9me2 marks at the nuclear periphery in P. megistus cells, differing from data for T. infestans, is suggested to reflect differences in the interaction of lamina-associated chromatin domains with the nuclear lamina, methyl-transferase modulation and/or association with the last DNA endoreplication step in 5th instar nymphs, which is a matter for further investigation.

Histone epigenetic marks in heterochromatin and euchromatin of the Chagas' disease vector, Triatoma infestans.

Triatoma infestans, a vector of Chagas' disease, shows several particular cell biology characteristics, including the presence of conspicuous heterochromatic bodies (chromocenters) where DNA methylation has not been previously detected. Whether histone modifications contribute to the condensed state of these bodies has not yet been studied. Here, we investigated epigenetic modifications of histones H3 and H4 and presence of the non-histone heterochromatin protein (HP1-α) in the chromocenters and euchromatin of T. infestans cell nuclei, using immunocytochemistry. The effect of different concentrations of the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (NaBt) on chromocenter condensation was visually examined; in VPA-treated specimens, this effect was also analyzed by image analysis. Trimethylated H3K9 signals, which were revealed in chromocenter and non-chromocenter areas, were strongest in chromocenters, whereas selected acetylated histone marks and mono- and dimethylated H3K9 and H4K20 signals were detected only in euchromatin. Weak trimethylated H4K20 signals and variable distribution of HP1-α were detected in chromocenters of part of the cellular population analyzed. Although specific VPA and NaBt treatment conditions affected the heterochromatin condensation pattern, they did not induce a decrease in survival and molting rates of the T. infestans nymphs. The VPA-induced chromatin remodeling was not accompanied by induction of H3K9 acetylation in chromocenters. Present findings regarding histone modifications and effects following VPA or NaBt treatments did not yet solve the question of which factors are responsible for maintenance of the condensed state of chromocenters in T. infestans. A possibility requiring further investigation remains on histone methylation marks and/or non-histone proteins.

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (Hemiptera, Reduviidae).

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug.

Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA(3)/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.

On an acute case of Chagas disease in a region under vector control in the state of São Paulo, Brazil.

No vector transmitted cases of Chagas disease had been notified in the state of São Paulo since the 1970s. However, in March, 2006, the death of a six-year-old boy from the municipality of Itaporanga was notified to the Center for Epidemiological Survey of the São Paulo State Health Secretariat: an autochthonous case of acute Chagas disease. The postmortem histopathological examination performed in the Hospital das Clínicas of the Botucatu School of Medicine confirmed the diagnosis. Reference to hospital records, consultation with the health professionals involved in the case and interviews with members of the patient's family supplied the basis for this study. We investigated parasite route of transmission, probable local reservoirs and vectors. No further human cases of acute Chagas disease were diagnosed. No locally captured vectors or reservoirs were found infected with Trypanosoma cruzi. Alternative transmission hypotheses - such as the possible ingestion of foods contaminated with vector excreta - are discussed, as well as the need to keep previously endemic regions and infested houses under close surveillance. Clinicians should give due attention to such signs as uni- or bilateral palpebral edema, cardiac failure, myocarditis, pericarditis, anasarca and atypical signs of nephrotic syndrome or nephritis and consider the diagnostic hypothesis of Chagas disease.

On an acute case of Chagas disease in a region under vector control in the state of São Paulo, Brazil / Sobre caso de doença de Chagas aguda em região de vetores controlados no Estado de São Paulo, Brasil

No vector transmitted cases of Chagas disease had been notified in the state of São Paulo since the 1970s. However, in March, 2006, the death of a six-year-old boy from the municipality of Itaporanga was notified to the Center for Epidemiological Survey of the São Paulo State Health Secretariat: an autochthonous case of acute Chagas disease. The postmortem histopathological examination performed in the Hospital das Clínicas of the Botucatu School of Medicine confirmed the diagnosis. Reference to hospital records, consultation with the health professionals involved in the case and interviews with members of the patient's family supplied the basis for this study. We investigated parasite route of transmission, probable local reservoirs and vectors. No further human cases of acute Chagas disease were diagnosed. No locally captured vectors or reservoirs were found infected with Trypanosoma cruzi. Alternative transmission hypotheses - such as the possible ingestion of foods contaminated with vector excreta - are discussed, as well as the need to keep previously endemic regions and infested houses under close surveillance. Clinicians should give due attention to such signs as uni- or bilateral palpebral edema, cardiac failure, myocarditis, pericarditis, anasarca and atypical signs of nephrotic syndrome or nephritis and consider the diagnostic hypothesis of Chagas disease.

Desde a década de 1970 não se notificavam casos autóctones de doença de Chagas aguda em São Paulo. Em março de 2006 a Vigilância Epidemiológica registrou óbito por doença de Chagas aguda, em Itaporanga, de paciente de seis anos de idade. Exame histopatológico post mortem realizado no Hospital das Clínicas da Faculdade de Medicina de Botucatu confirmou o diagnóstico. Consultamos prontuários de hospitais e entrevistamos profissionais de saúde envolvidos além de familiares do paciente. Descrevemos medidas adotadas in loco para identificar a via de transmissão, reservatórios e vetores. Discutimos as possíveis fontes de infecção. Na região não foram identificados outros casos humanos, vetores ou reservatórios vertebrados infectados por Trypanosoma cruzi. Salientamos a importância de manter a vigilância, mesmo em áreas onde a transmissão de doença de Chagas está interrompida e naquelas ainda infestadas por triatomíneos. Deve-se admitir a hipótese diagnóstica de doença de Chagas quando observados: edema palpebral (uni ou bilateral), insuficiência cardíaca, miocardite, pericardite, anasarca, quadros similares aos de síndrome nefrótica ou glomerulonefrite sem causas outras aparentes, em pacientes com dados epidemiológicos positivos. Encontro, mesmo em raras ocasiões, de triatomíneos na região ou ainda contato com alimento contaminável com formas infectantes de T. cruzi.

Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect, Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae)

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40ºC, 1 h) or cold (5 or 0ºC, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0ºC, respectively, after 8, 18, 24 and 72 h at 28ºC (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0ºC were more effective than the temperatures of 35 and 5ºC in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes

Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect, Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae).

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40 degrees C, 1 h) or cold (5 or 0 degrees C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0 degrees C, respectively, after 8, 18, 24 and 72 h at 28 degrees C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0 degrees C were more effective than the temperatures of 35 and 5 degrees C in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.

Triatoma infestans em área sob vigilância entomológica para doença de Chagas, Estado de Säo Paulo, Brasil / Triatoma infestans in area under entomological surveillance for Chagas'diseases in Säo Paulo State, Brazil

Atendendo a notificação de encontro, por habitantes da Fazenda Paraíso, área rural do Município de Paulínea, Estado de São Paulo, de um exemplar alado de triatomíneo, procedeu-se a uma investigaçao epidemiológica em que capturaram-se 109 exemplares de triatoma infestans em focos situados em construçoes peridomiciliares. As condiçóes locais favoreciam a colonizaçao por triatomíneos: grande número de construçoes peridomiciliares abandonadas, habitadas por pombos e pardais, fornecedores de farto alimento para os hemípteros. Eliminaram-se os focos por meio de controle mecânico dos ninhos dos pássaros. Borrifaram-se, com inseticida de ação residual todas as unidades domiciliares da regiao. Desde que persistam as condições para a instalacao de focos de triatomíneos nessa localidade, é obrigatória a implementaçao de açoes de vigilancia. Entretanto, os indicadores entomológicos e sorológicos sugerem nao ser preocupante a situaçao atual. Destaca-se aqui a importância da notificação triatomínica para a detecçao de focos de triatomíneos, particularmente os de Triatoma infestans

Estudo epidemiológico de fontes de alimentaçäo sangüínea dos triatomíneos da fazenda Aroeira (Catolé do Rocha, Paraíba) e circunvizinhanças / Epidemiologic study of the sources of blood feeding of the triatominae of the Aroeira farm (Catolé do Rocha, Paraíba) and neighboring localities

Analisamos a fonte de alimentaçäo sangüínea de 94 triatomíneos (51 Triatoma brasiliensis, 42 T. pseudomaculata e um Rhodnius neglectus) da Fazenda Aroeira, no Município de Catolé di Rocha, no Estado da Paraíba, e de quatro localidades próximas. Observamos postividade para marsupial, homem e ave, respectivamente, de 39,7, 6,2 e 23,0 por cento. Três insetos da Fazenda Aroeira, positivos para Trypanosoma cruzi, foram positivos para marsupial, só um deles também para outro mamífero. Os marsupiais säo a mais importante fonte de T. cruzi para os insetos da área e estes têm pouco contato com o homem