Feulgen staining in malpighian tubules of meliponid bees: a methodological contribution

Whole-mounted Malpighian tubules of larvae from two meliponid bee species fixed in acetic acid-ethanol showed apositive cytoplasmic staining with Schiff reagent when submitted to the Feulgen reaction in which acid hydrolysiswas done in 4 M HCl at room temperature. The ability of various treatments applied before the Feulgen acid hydrolysisto abolish this cytoplasmic staining was examined. The aldehyde groups of phospholipids present in the cytoplasm ofthe Malpighian tubules were blocked or removed by sequential treatment with 5% sodium borohydride and acetonechloroform(1:1, v/v) for 15 min each prior to HCl hydrolysis. This treatment is recommended in order to abolish thecytoplasmic (plasmal) reaction and to allow the reliable quantification of DNA by the Feulgen reaction and thediscrimination of nuclear phenotypes in the Malpighian tubules of meliponid bees.

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

Changes in nuclear phenotype frequencies following sequential cold shocks in Triatoma infestans (Hemiptera, Reduviidae)

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 Säo 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

Effect of sequential cold shocks on survival and molting rate in Triatoma infestans Klug

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

Changes in nuclear phenotypes following cold shock in Panstrongylus megistus (Burmeister)

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 0o.C, 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 percent 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

Nuclear phenotype changes after heat shock in Panstrongylus megistus (Burmeister)

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.

Morphological dimorphism in the Y chromosome of "pé-duro" cattle in the brazilian state of Piauí

Um plantel de touros "pé-duro", consistindo de 75 animais do núcleo da Embrapa envolvido com a preservaçäo desse gado no Estado do Piauí, foi examinado quanto à morfologia do seu cromossomo Y, bem como em relaçäo a elementos da genitália externa e outras características fenotípicas dos machos. O objetivo era investigar a contaminaçäo 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 contaminaçäo racial por gado zebuíno, foram detectadas no rebanho "pé-duro" mantido no núcleo da Embrapa. Outras características fenotípicas analisadas que podem informar sobre a contaminaçäo racial aparente näo foram associadas ao tipo de cromossomo Y. A detecçäo de contaminaçäo racial no estoque de touros "pé-duro" aqui analisado näo invalida os esforços da Embrapa em preservar este gado de importância regional para o Nordeste do Brasil, porém quase extinto.

Changes in the nuclear phenotypes of Triatoma infestans KLug, induced by thermal shocks*

Os fenótipos nucleares de células epiteliais dos túbulos de Malphigi de ninfas de 4§ estadio de Triatoma infestans Klug submetidas a choques de temperatura a 0 gradeC e 40 gradeC por 1 e 12 horas foram investigados imediatamente após o estress e 3 e 30 dias após o mesmo. Além dos fenótipos usualmente descritos para os espécimes controle, nucleos pienóticos, gigantes e vacuolizados, bem como núcleos com uma aparente descompactaçäo da cromatina do cromocentro foram detectados nos espécimes-teste. Admite-se que um decréscimo no número de núcleos a curto ou longo prazo após os choques de temperatura seja contribuído näo só pela degeneraçäo das células que apresentam núcleos vacuolizados e picnóticos, mas também por fusäo nuclear. Supöe-se que os núcleos gigantes, que apareceram com predominância nos espécimes submetidos ao choque hipertérmico e, ao mesmo tempo, a presença de hsp, desempenhem papel na manutençäo da sobrevivência do inseto nesta condiçäo experimental específica. Embora muitas das características nucleares encontradas nos espécimes submetidos aos choques de temperatura tenham sido também descritas após açäo de outros agentes estressantes, o choque hipertérmico foi o mais efetivo na promoçäo da aparente descompactaçäo da heterocromatina do crfomocentro. Com base nos tipos e frequências dos núcleos encontrados após choque hipertérmico e queda na sobrevivência do inseto sob tal circunstância, considera-se que, se existem substâncias crioprotetoras em T. infestans, estas näo seriam eficientes para proteger o inseto de um choque hipotérmico relativamente longo

Sobrevivência e ocorrência de muda em Triatoma infestans Klug (Hemiptera, Reduviidae) após choque de temperatura / Survival and occurrence of molting in Triatoma infestans Klug (Hemiptera, Reduviidae) after temperature shock

A sobrevivência e a ocorrência de mudas em espécies de Triatoma infestans foram estudadas 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 duraçäo, fase de desenvolvimento e sexo dos espécimes. Dentre as situaçöes experimentais utilizadas, o choque a 0§C por 12 h pareceu produzir a açäo mais deletéria, porém choques a 40§C e 0§

Feulgen-DNA content evaluation and image analysis of NIH 3T3 cells transfosmed ncf-7 cell DNA

O conteúdo Feulgen-DNA, bem como os tamanhos e fenótipos nucleares de células NIH/3T3 transformadas por transfecçäo com o DNA genômico contendo oncogene N-ras decélulas MCF-7, foram estudados por microespectrofotometria de varredura e análise de imagem e comparados aos exibidos quando a transforamaçäo é obtida por transfecçäo com o DNA do oncogene c-H-ras de células T24. Foram encontrados níveis superiores de condensaçäo cromatínica afetando a eucromatina, o que em seus aspectos gerais caracteriza uma situaçäo semelhante áquela obtida quando a transformaçäo é mediada pelo DNA do oncogene c-H-ras de células T24. Supöe-se que a condensaçäo cromatinica induzida esteja associada a uma repressäo de genes tornados näo disponíveis para transcriçäo e/ou represente um efeito topológico sobre a geometria de complexo nucleoprotéicos, induzido por açöes próximas ou distantes ao longo da molécula de DNA. Em parte das células transformadas pela transfecçäo com o DNA da células MCF-7 a condensaçäo cromatínica excedeu aquela das células transformadas por transfecçäo com o oncogene c-H-ras. Admite-se que isto seja uma diferente resposta clonal de células NH/3T3 após transfecçäo com oncogenes ras ou seja causado pela composiçäo do DNA transfectado, o qual compreendia o genoma total de células MCF-7

Nuclear ultrastructure of malpighian tubule cells in Triatoma infestans (Hemiptera, Reduviidae) under conditions of full nourishment and starvation

A ultra-estrutura de núcleos, com ênfase complexo cromocentro-nucléolo, foi estudada em células dos túbulos de Malpighi de espécimes de Triatoma infestans bem alimentados e sob jejum. Foram observadas, pelo menos em parte dos núcleos dos insetos em jejum, alteraçöes envolvendo o aparecimento de grânulos de paricromatina na zona superficial dos cromocentros näo em contato com componentes nucleares, desaparecimento total ou parcial dos elementos do nucleolonema e decréscimo na elétron-opacidade de alguns cromocentros. Admite-se que esta última esteja associada à descompactaçäo de heterocromatina induzida pelo "stress" e sugerida anteriormente com análise de imagem. O aspecto ultraestrutural dos núcleos 24 horas após os insetos em jejum se alimentarem praticamente näo difere daquele dos controles bem alimentados. Pequenos grânulos näo descorados com o método do EDTA, descrito como corando preferncialmente RNA, foram encontrados no interior de cromocentros de insetos bem alimentados ou naqueles que se alimentaram após o jejum. Estas áreas coradas säo admitidas como regiöes dos cromocentros contendo cromatina menos firmemente empacotada e säo especuladas como possíveis sítios com algum potencial transcritivo