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°C, 1 h) or cold (5 or 0°C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0°C, respectively, after 8, 18, 24 and 72 h at 28°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 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.

The nuclear phenotypes of Malpighian tubule cells in fifth instar nymphs of Triatoma infestans, one of the most important vectors of Chagas disease, were studied following sequential shocks at 0ºC, separated by intervals of 8 h and 24 h at 30ºC, under conditions of moderate fasting and full nourishment. The insects pertained to colonies reared in the laboratory and originated from domestic specimens collected in the Brazilian states of São Paulo (north) and Minas Gerais (south). Since nuclear phenotypes in this species are affected by single cold shocks, it was expected that these phenotypes could also be changed by sequential shocks. Nuclear phenotypes indicative of mechanisms of cell survival (nuclear fusion and heterochromatin decondensation) and cell death (apoptosis and necrosis) were observed concomitantly in all the conditions tested. Nuclear fusion and heterochromatin decondensation were not found relevant for the presumed acquisition of the cold-hardening response in T. infestans. The decreased frequency of apoptosis and necrosis following sequential cold shocks including under fasting conditions, indicated that tolerance to sequential cold shocks occurred in T. infestans of the mentioned origin.

The survival and molting incidence in Triatoma infestans, a vector of Chagas disease, were investigated following sequential shocks at 0ºC in fifth instar nymphs under moderate fasting and full nutritional conditions. The shocks were separated by intervals of 8 h and 24 h at 30ºC. The results indicated that in terms of insect survival, T. infestans is tolerant to a single cold shock at 0ºC even for 12 h, or to sequential cold shocks, regardless of the nutritional state of the specimens. In terms of molting rate, fasting enhanced the tolerance to sequential cold shocks, but did not exceed the tolerance acquired by fully-nourished specimens, except when cold shocks were separated by an 8 h interval at 30ºC. The protective action elicited by fasting was assumed to be additive to that induced by a single mild cold shock or sequential cold shocks. The cold-tolerance response of T. infestans may have favoured its survival in areas of South America with low temperatures, even considering that this species is predominantly associated with human habitats.

The nuclear phenotypes of Malpighian tubule epithelial cells of 5th instar male nymphs of the blood-sucking insect Panstrongylus megistus were studied immediately after a short (1 h) cold shock at 0ºC, and 10 and 30 days later. The objective was to compare the responses to a cold shock with those known to occur after hyperthermia in order to provide insight into the cellular effect of cold in this species. Nuclei which usually exhibited a conspicuous Y chromosome chromocenter were the most frequent phenotype in control and treated specimens. Phenotypes in which the heterochromatin was unravelled, or in which there was nuclear fusion or cell death were more abundant in the shocked specimens. Most of the changes detected have also been found in heat-shocked nymphs, except for nuclear fusion which generates giant nuclei and which appeared to be less effective or necessary than that elicited after heat shock. Since other studies showed that a short cold shock does not affect the survival of more than 14% of 5th instar nymphs of P. megistus with domestic habit and can induce tolerance to a prolonged cold shock, heat shock proteins proteins are probably the best candidates for effective protection of the cells and the insects from drastic damage caused by low temperature shocks.

Uma vez que em Triatoma infestans a sobrevivência de células somáticas e dos próprios espécimes é afetada por choques de temperatura, foi estudada a indução de morte celular em células da linhagem germinativa masculina de ninfas de 5o. estadio 7 e 30 dias após choque de 1 h a 40ºC. Os preparados foram submetidos à reação de Feulgen e ao teste imunocitoquímico TUNEL. Foi encontrada apoptose induzida pelo choque de temperatura, sendo que respostas positivas ao TUNEL foram vistas na cromatina e em cromossomos de espermatogônias e em espermátides e espermatozóides. As espermatogônias foram afetadas principalmente 7 dias após o choque, enquanto algumas espermátides e espermatozóides exibiram fragmentação de DNA apenas 30 dias após o choque. Admite-se que a velocidade com a qual avança o processo de morte celular varie nessas células. Diferenciação celular não é impedida em parte das células afetadas, permitindo que a fragmentação de seu DNA ocorra mais tardiamente, inclusive no fim da espermatogênese.

The survival of Triatoma infestans and of somatic cells from this species is affected by heat shock. In this study, we examined the cell death responses of male germ line cells from 5th instar nymphs 7 and 30 days after heat shock exposure (40ºC, 1 h). The preparations were stained by the Feulgen reaction and the TUNEL immunocytochemical assay. Apoptosis was elicited by heat shock, with positive TUNEL responses in spermatogonial chromatin and chromosomes, spermatids and sperm cells. Spermatogonia were most affected seven days after the shock whereas some spermatids and sperm cells exhibited DNA fragmentation only thirty days after heat shock. The rate of cell death varied among the cells. In some cases, cellular differentiation was unaffected by heat shock, with DNA fragmentation occurring towards the end of spermatogenesis.

The nuclear phenotypes of Malpighian tubule epithelial cells of male nymphs of the blood-sucking insect, Panstrongylus megistus, subjected to short- and long-duration heat shocks at 40ºC were analyzed immediately after the shock and 10 and 30 days later. Normal nuclei with a usual heterochromatic body as well as phenotypes indicative of survival (unravelled heterochromatin, giants) and death (apoptosis, necrosis) responses were observed in control and treated specimens. However, all nuclear phenotypes, except the normal ones, were more frequent in shocked specimens. Similarly altered phenotypes have also been reported in Triatoma infestans following heat shock, although at different frequencies. The frequency of the various nuclear phenotypes observed in this study suggests that the forms of cell survival observed were not sufficient or efficient enough to protect all of the Malpighian tubule cells from the deleterious effects of stress. In agreement with studies on P. megistus survival following heat shock, only long-duration shock produced strongly deleterious effects.

Análise de imagem de núcleos de fibroblastos V79 após três dias em cultura foi realizada em preparados submetidos à reação de Feulgen para discriminar células suspeitas de estarem em fases precoces da apoptose daquelas comprovadamente apoptóticas. Parâmetros tais como conteúdo de Feulgen-DNA, grau de condensação cromatínica e área nuclear foram estudados em associação com a morfologia estabelecida em termos visuais. Os fibroblastos foram inicialmente plaqueados numa densidade de 10(5) células/ml e encubados sob condições ótimas de cultura sem subcultura. Com a confluência, as células sofreram apoptose pela inibição por contato. A análise de imagem revelou três fenótipos nucleares definidos quanto a características morfológicas e níveis de condensação cromatínica. Foi detectado decréscimo no conteúdo de Feulgen-DNA das células apoptóticas mas também nas células suspeitas de apoptose. Admite-se que este decréscimo indique perda de DNA. A análise de imagem pode ser, portanto, uma ferramenta útil na discriminação de células mesmo em fases precoces da apoptose.

Nuclear image analysis of Feulgen-stained V79 fibroblasts after three days in culture was used to discriminate apoptotic cells and cells suspected to be undergoing apoptosis from control cells based on parameters such as the Feulgen-DNA content, degree of chromatin condensation and nuclear areas, in association with visual morphology. The fibroblasts were initially plated at a density of 10(5) cells/ml and incubated under optimal culture conditions without subculturing. Following confluency, the cells underwent contact inhibition apoptosis. Image analysis revealed three nuclear phenotypes which were defined in terms of their morphological characteristics and levels of chromatin condensation. A decrease in the amount of Feulgen-DNA was detected in apoptotic cells and in cells suspected of undergoing apoptosis. This decrease was assumed to indicate DNA loss. Image analysis procedures may therefore provide a useful tool for discriminating cells in the early stages of apoptosis.

Um plantel de touros "pé-duro", consistindo de 75 animais do núcleo da Embrapa envolvido com a preservação desse gado no Estado do Piauí, foi examinado quanto à morfologia do seu cromossomo Y, bem como em relação a elementos da genitália externa e outras características fenotípicas dos machos. O objetivo era investigar a contaminação racial por animais zebuínos (Bos taurus indicus) num gado bovino que tem sido considerado mais próximo de sua origem européia (Bos taurus taurus). Tanto a forma submetacêntrica quanto a forma acrocêntrica do cromossomo Y, típicas das sub-espécies B. t. taurus e B. t. indicus, respectivamente, bem como maior bainha prepucial nos espécimes portadores do cromossomo Y acrocêntrico, indicativa de contaminação racial por gado zebuíno, foram detectadas no rebanho "pé-duro" mantido no núcleo da Embrapa. Outras características fenotípicas analisadas que podem informar sobre a contaminação racial aparente não foram associadas ao tipo de cromossomo Y. A detecção de contaminação racial no estoque de touros "pé-duro" aqui analisado não invalida os esforços da Embrapa em preservar este gado de importância regional para o Nordeste do Brasil, porém quase extinto.

"Pé-duro" (hard foot) is a rare breed of beef cattle of European (Bos taurus taurus) origin, originated in northern and northeastern Brazil. Y chromosome morphology, outer genital elements and other phenotypic characteristics were examined in 75 "pé-duro" bulls from the Empresa Brasileira de Pesquisa Agropecuária (Embrapa) herd in the Brazilian State of Piauí. The purpose was to investigate possible racial contamination with Zebu animals (Bos taurus indicus) in a cattle that has been considered closest to its European origin (B. t. taurus). The presence of both submetacentric and acrocentric Y chromosomes, typical of B. t. taurus and B. t. indicus, respectively, and the larger preputial sheath in bulls with an acrocentric Y chromosome indicated racial contamination of the "pé-duro" herd with Zebu cattle. Phenotypic parameters involving horn, dewlap, ear, chamfer, and coat color characteristics, indicative of apparent racial contamination, were not associated with acrocentric Y chromosome.

Survival and molting incidence were studied after heat (40°C) and cold (0°C) shocks in specimens of Panstrongylus megistus with the aim of establishing its response to temperature stress under laboratory rearing conditions and to understand occasional changes in the biological characteristics of specimens captured in nature. The response to the thermal shocks was found to vary as a function of the temperature and duration of the shock, developmental phase and sex of the specimens, and in certain cases, the insect habit and nourishment conditions. P. megistus specimens were found to be less resistant to the heat shock assay than Triatoma infestans, another reduviid species. The short cold shock affected survival of P. megistus more than did the heat shock, survival of fully-nourished specimens being preferential. The response of adults to the short cold shock was affected by sex, males being generally less resistant. The insect sylvatic habit was found to seldom affect the thermal shock response established for specimens with domestic habit. A decrease in molting frequency and sometimes a slowdown of the molting rate were found after the short heat and cold shocks, possibly promoted by change in hormonal balance, and differing from patterns reported for T. infestans. The results indicate that no generalization should be made for different reduviid species in terms of the effects of temperature shocks.

A ligação de moléculas de azul de toluidina a grupamentos fosfatos de ácido nucléico em condições de competição com íons Mg2+ foi estudada em regiões de cromossomos politênicos de Trichosia pubescens que diferem em composição e organização (áreas ricas em RNA, pufes de RNA e DNA, heterocromatina). O objetivo foi encontrar variabilidade na concentração de Mg2+ na qual a metacromasia fosse completamente abolida (= concentração crítica de eletrólitos; CEC) e que refletisse diferenças ao nível dos complexos nucleoprotéicos em regiões cromossômicas e fases do desenvolvimento particulares. Embora valores altos de CEC tenham sido encontrados na cromatina altamente empacotada, como nas zonas heterocromáticas, os valores de CEC para outras regiões mostraram-se afetados não apenas pela disponibilidade e proximidade de fosfatos do DNA mas também pela riqueza em RNA e outros fatores. Entre estes, mudanças na geometria e no estado de empacotamento do DNA foram consideradas para explicar valores aumentados após remoção de RNA em pufes de DNA e zonas ricas em RNA. Baseando-se em valores de CEC foi sugerido que possam ocorrer alterações ao nível dos complexos nucleoprotéicos em pufes antes que estes sejam detectáveis morfologicamente como tal. Os valores de CEC nos cromossomos politênicos revelaram-se, pois, altamente variáveis com as variações em composição e organização nucleoprotéica do substrato. Contudo, uma vez que um raciocínio complexo e aparentemente contraditório tem que ser utilizado para a interpretação de parte dos resultados, a CEC não é recomendável como ferramenta para estudos extensivos e comparativos neste modelo em particular.

The binding of toluidine blue molecules to nucleic acid phosphates under conditions of competition with Mg2+ ions was studied in Trichosia pubescens polytene chromosome regions differing in composition and organization (RNA-rich areas and puffs, DNA puffs, heterochromatin). The aim was to find variability in the Mg2+ concentration at which metachromasy was completely prevented (= critical electrolyte concentration; CEC) and that could reflect differences at the level of nucleoprotein complexes in particular chromosome regions and developmental phases. Although high CEC values have been found in tightly packed chromatin such as that of heterochromatin zones, the CEC values for the other regions have proven to be affected not only by availability and proximity of DNA phosphates but also by RNA richness and other factors. Among these, changes in DNA geometry and packing state have been assumed for explaining increased values after RNA removal in DNA puffs and RNA-rich zones. Based on CEC values it has been suggested that alterations at the level of nucleoprotein complexes may occur in puffs before they are morphologically detectable. CEC results for polytene chromosomes were thus revealed to vary considerably with slight variations in nucleoprotein composition and organization. However, since a complex and apparently contradictory rationale has to be used for explaining part of the results, CEC is not recommended as a useful tool for extensive and comparative studies of this particular model.

A sobrevivência e a ocorrência de mudas em espécimes de Triatoma infestans foram estudados num período de 30 dias após choques de temperatura. Foi demonstrado que choques hipertérmico e hipotérmico interferem nesses processos, na dependência da temperatura do choque, tempo de sua duração, fase de desenvolvimento e sexo dos espécimes. Dentre as situações experimentais utilizadas, o choque a 0°C por 12 h pareceu produzir a ação mais deletéria, porém choques a 40°C e 0°C mesmo por uma hora são admitidos como afetando o desenvolvimento hormonal que controla a muda. Casos de aumento de sobrevivência pós-choque são sugeridos como tendo sido favorecidos por ação de proteínas de choque térmico.

Survival and molting occurrence were studied in specimens of Triatoma infestans over 30 days after temperature shocks. Hyperthermal and hypotermal shocks could be found to affect both survival and molting incidence as a function of temperature and period of the developement phase and sex of the specimens. Considering the various test conditions, the shock at 0°C for 12 h was found to elicit the most deleterious effect, whereas shocks at 40°C and 0°C even for 1 h are interpreted as affecting the hormonal balance which controls molting. Cases of a rise in post-shock survival are suggested to have been favored by heat-shock protein action.

A sobrevivência e a ocorrência de mudas em espécimes de Triatoma infestans foram estudados num período de 30 dias após choques de temperatura. Foi demonstrado que choques hipertérmico e hipotérmico interferem nesses processos, na dependência da temperatura do choque, tempo de sua duração, fase de desenvolvimento e sexo dos espécimes. Dentre as situações experimentais utilizadas, o choque a 0°C por 12 h pareceu produzir a ação mais deletéria, porém choques a 40°C e 0°C mesmo por uma hora são admitidos como afetando o desenvolvimento hormonal que controla a muda. Casos de aumento de sobrevivência pós-choque são sugeridos como tendo sido favorecidos por ação de proteínas de choque térmico.

Survival and molting occurrence were studied in specimens of Triatoma infestans over 30 days after temperature shocks. Hyperthermal and hypotermal shocks could be found to affect both survival and molting incidence as a function of temperature and period of the developement phase and sex of the specimens. Considering the various test conditions, the shock at 0°C for 12 h was found to elicit the most deleterious effect, whereas shocks at 40°C and 0°C even for 1 h are interpreted as affecting the hormonal balance which controls molting. Cases of a rise in post-shock survival are suggested to have been favored by heat-shock protein action.

Com o objetivo de se estudar a ação da oxamniquine, uma droga utilizada no tratamento da esquistossomose, sobre a cromatina de núcleos de células animais, foram estudados os padrões de basofilia e anisotropia nucleares em hepatócitos, em células do músculo cardíaco e em linfócitos de camundongos adultos jovens. A oxamniquine foi administrada por via oral (436 mg/kg) e preparados foram obtidos após diversos tempos de fornecimento da droga aos animais. Nos núcleos corados com azul de toluidina a pH 4,0, após digestão com RNAse, não se encontrou diferença quanto aos padrões de basofilia e anisotropia comparando-se animais tratados com controles. Demonstrou se assim que as moléculas de oxamniquine não se alojam no interior da dupla hélice do DNA, não alteram a sua conformação helicoidal nem se ligam aos grupos fosfatos livres desta macromolécula, diferindo, portanto, da atuação de outro esquistossomicida, o hycanthone.

With the aim of evaluating the effect of oxamniquine, a drug used in the clinical trials of schistosomiasis mansoni, on chromatin of animal cell nuclei, the patterns of nuclear basophilia and anisotropy were determined for hepatocytes, cardiac miocytes, and lymphocytes of young adult mice. The animals received the oxamniquine orally in a single dose of 436 mg/kg. Cell preparations were obtained at various times after the administration of the drug. The nuclei were subjected to RNAse digestion and stained with a toluidine blue solution at pH 4.0. No difference was found when comparing the patterns of nuclear basophilia and anisotropy of treated animals with those of untreated controls. It was demonstrated that the oxamniquine molecules do not penetrate the interior of the DNA double helix, neither do they alter the helical conformation of this nucleic acid. Furthermore, the oxamniquine molecules do not attach themselves to the free phosphate groups of the DNA. Therefore, this drug does not affect the DNA of the animal chromatin, differing from another schistosomicide, hycanthone.

Nos tubos de Malpighi de Triatoma infestans Klug e de Panstrongylus megistus Burmeister foram encontradas estruturas víricas distribuídas no citoplasma, no interior de citolissomos ou outros tipos de glóbulos, formando às vezes arranjos para cristalinos envolvidos por membrana, e entre plasmalemas de células contíguas. Outros tecidos dos mesmos insetos apresentam arranjos paracristalinos desses virus. A análise citoquímica ao nível da microscopia eletrônica indica serem estes virus compostos de RNP. Não são encontradas alterações morfológicas drásticas nos tecidos infectados. Admite-se que tais virus tenham sido ingeridos durante uma alimentação de sangue infectado de ave.

Viral particles distributed within cytolysomes or other cytoplasmic globule types, giving rise to membrane enclosed paracrystalline arrays, and between membranes of contiguous cells, were found in the Malpighian tubes of Triatoma infestans Klug and Panstrongylus megistus Burmeister. Other tissues of the same insects also display paracrystalline arrays of these viruses. A RNP composition is assumed for the viruses based on their response to cytochemical methods at the EM level. Drastic morphological changes were not found in the injected tissues. This infection assumed to be promoted by ingestion of a bird infected blood meal.

Nos tubos de Malpighi de Triatoma infestans Klug e de Panstrongylus megistus Burmeister foram encontradas estruturas víricas distribuídas no citoplasma, no interior de citolissomos ou outros tipos de glóbulos, formando às vezes arranjos para cristalinos envolvidos por membrana, e entre plasmalemas de células contíguas. Outros tecidos dos mesmos insetos apresentam arranjos paracristalinos desses virus. A análise citoquímica ao nível da microscopia eletrônica indica serem estes virus compostos de RNP. Não são encontradas alterações morfológicas drásticas nos tecidos infectados. Admite-se que tais virus tenham sido ingeridos durante uma alimentação de sangue infectado de ave.

Viral particles distributed within cytolysomes or other cytoplasmic globule types, giving rise to membrane enclosed paracrystalline arrays, and between membranes of contiguous cells, were found in the Malpighian tubes of Triatoma infestans Klug and Panstrongylus megistus Burmeister. Other tissues of the same insects also display paracrystalline arrays of these viruses. A RNP composition is assumed for the viruses based on their response to cytochemical methods at the EM level. Drastic morphological changes were not found in the injected tissues. This infection assumed to be promoted by ingestion of a bird infected blood meal.