Ampullae infection of the malpighian tubules of Bombyx mori by Alphabaculovirus / Infecção de células da ampola dos túbulos de malpighi de Bombyx mori pelo Alphabaculovirus

Alphabaculovirus is a genus of the entomopathogenic virus, whose species Bombyx mori nucleopolyhedrovirus (BmNPV) infects the silkworm, Bombyx mori, which is an important insect in the sericulture industry. A geographic isolate of BmNPV was identified in the state of Paraná, Brazil. It was infecting B. mori larvae and various organs and target tissues were identified, however, there was no information about the infection of Malpighian tubules (MT). The MT comprises the excretory system of B. mori and acts in the elimination of toxic substances and in hydroelectrolytic homeostasis. Thus, the present study examined the susceptibility and cytopathology of B. mori MT to BmNPV. To this end, hybrid fifth instar larvae were inoculated with a virus suspension at different days post-inoculation (dpi). MT segments were collected and divided into the ampullae, proximal, medial and distal regions. These were processed for light microscopy and transmission electron analysis. The MT regions revealed differences in susceptibility to BmNPV and the ampullae in its transition area was infected from the sixth dpi; the other regions did not reveal any evidence of infection. The transition area of the ampullae has not been previously described in Lepidoptera and its cytopathology revealed a hypertrophic nucleus with viroplasm, followed by the formation and development of viral polyhedra, which are common characteristics of infections by Alphabaculovirus. Thus, infection of the ampullae of the MT of B. mori by BmNPV, together with other known targets, compromises the metabolic balance of the insect, which results in consequences for silk production and damage to the sericulture sector.

Alphabaculovirus é um gênero de vírus entomopatogênico, cuja espécie Bombyx mori nucleopolyhedrovirus (BmNPV) infecta o bicho-da-seda, Bombyx mori, inseto importante na indústria sericícola. Um isolado geográfico do BmNPV foi identificado no estado do Paraná, Brasil, infectando lagartas de B. mori e vários órgãos e tecidos alvos foram identificados; entretanto, não há informações sobre a infecção do túbulo de Malpighi (TM). O TM compõe o sistema excretor de B. mori, atuando na eliminação de substâncias tóxicas e na homeostase hidroeletrolítica. Assim, o presente estudo, analisou a susceptibilidade e a citopatologia do TM de B. mori ao BmNPV. Para tanto, lagartas híbridas de 5° instar foram inoculadas com uma suspensão viral e em diferentes dias pós-inoculação (dpi), segmentos do TM foram coletados e subdivididos nas regiões da ampola, proximal, média e distal; sendo processados para análises em microscopias de luz e eletrônica de transmissão. As regiões do TM revelaram diferenças na susceptibilidade ao BmNPV e a ampola, na sua área de transição, foi infectada a partir do 6° dpi, já as demais regiões não revelaram quaisquer indícios de infecção. A área de transição da ampola, ainda não havia sido descrita em lepidópteros e sua citopatologia revelou núcleo hipertrófico com viroplasma, seguido da formação e desenvolvimento dos poliedros virais, características comuns das infecções pelo Alphabaculovirus. Assim, a infecção da ampola do TM de B. mori ao BmNPV, somada a de outros alvos conhecidos, compromete o equilíbrio metabólico do inseto, com consequências na produção de seda e prejuízos ao setor sericícola.

Prevalence and intensity of infection, metacyclogenesis and nuclear phenotypes in Panstrongylus megistus (Burmeister, 1835) after ingestion of Trypanosoma cruzi (Chagas, 1909) II and subjection to heat shock / Prevalência e intensidade de infecção, metaciclogênese e fenótipos nucleares em Panstrongylus megistus (Burmeister, 1835) após ingestão de Trypanosoma cruzi (Chagas, 1909) II e choque de temperatura

This study aimed to contribute to our knowledge of the parasite-vector interaction associated with Trypanosoma cruzi (Chagas, 1909) infection in Panstrongylus megistus (Burmeister, 1835), an important vector of Chagas' disease in Brazil. The prevalence and intensity of T. cruzi infection, the incidence of metacyclogenesis and the frequency of nuclear phenotypes in Malpighian tubules were investigated in nymphs of P. megistus, reared at 28 °C and subjected to heat shock (40 °C, 1 hour) two days after infection with T. cruzi II (Y strain). Following the 45-day post-infection period, the frequency of epimastigotes was much higher than that of trypomastigotes in both heat-shocked and non-shocked insects, and the prevalence of infection was not altered by heat shock. Fewer epimastigotes and trypomastigotes were found in the infected insects subjected to the heat shock, indicating that the multiplication and metacyclogenesis of the parasites were affected by the stress. In infected specimens heat shock promoted an increased frequency of cell nuclei with heterochromatin decondensation, a cell survival response to stress, and did not affect insect survival. The effects of infection and heat shock, especially on the multiplication and metacyclogenesis of T. cruzi, and the observed resistance to heat shock developed by P. megistus nymphs are suggestive that they should be considered when adequate conditions for rearing these infected insects in the laboratory are pursued.

O presente estudo teve como objetivo contribuir para o conhecimento sobre a interação parasita-vetor associada à infecção por Trypanosoma cruzi (Chagas, 1909) em Panstrongylus megistus (Burmeister, 1835), importante vetor da doença de Chagas no Brasil. A prevalência e a intensidade da infecção por T. cruzi, a incidência de metaciclogênese e a frequência de fenótipos nucleares presentes em túbulos de Malpighi foram investigadas em ninfas de P. megistus criadas à temperatura de 28 °C e submetidas a choque térmico por 1 hora a 40 °C, dois dias após alimentação em camundongos infectados com T. cruzi II (linhagem Y). A análise realizada 45 dias pós-infecção revelou que tanto nos insetos submetidos ao choque térmico como nos respectivos controles a frequência de epimastigotos se apresentava muito maior do que a dos tripomastigotos; a prevalência da infecção não foi alterada pelo choque térmico. Menos epimastigotos e tripomastigotos foram encontrados nos insetos submetidos ao choque térmico, indicando que a multiplicação e a metaciclogênese dos parasitas possam ser afetadas com o estresse. Nos espécimes infectados, o choque térmico induziu aumento na frequência de núcleos com descompactação da heterocromatina, uma resposta de sobrevivência celular ao estresse, e não afetou a sobrevivência propriamente dita do inseto. Os efeitos da infecção e do choque térmico, especialmente sobre a multiplicação e a metaciclogênese de T. cruzi, e a resistência ao choque térmico desenvolvidos pelas ninfas de P. megistus são indicativos de que devam ser considerados quando se buscam condições adequadas de criação em laboratório de insetos infectados.

Overexpression of kermit/dGIPC is associated with lethality in Drosophila melanogaster

Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3’ of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.

Analysis of nucleus activity in malpighian tubules of Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) larvae by critical electrolyte concentration

Diatraea saccharalis (Fabricius) has cryptonephridial type Malphigian tubules (MT). This type of MT is characterized by the penetration of the distal part of the MT into the external walls of the rectum, which is usually lined with a perinephral membrane. The MT is divided into three differentiated regions: proximal, middle and distal. In this study, our objective was to compare the nuclear activities of each one of the three regions of the D. saccharalis MT by using a nuclear basophilic technique and critical electrolyte concentration with a toluidine blue stain at pH 4.0. This method allows differentiation of DNA/protein complexes in in situ and in vitro chromatin. MT chromatin structure in D. saccharalis is variable. Fifth instars have a more decondensed chromatin than fourth instars. The distal tubule region was the most decondensed region of the MT. Our data show an elevated genetic activity of the MT in the pre-metamorphosis period. The distal region of the MT has the highest observed activity, which may be associated with the re-absorption of useful components and the excretion of waste materials.

Rhodnius prolixus Malpighian tubules and control of diuresis by neurohormones

Rhodnius prolixus Malpighian tubules (MTs) are a good model for fluid and ion secretion studies in view of the dramatic postprandial diuresis, which follows its massive blood meals. Ingestion of a blood meal equals to 10-12 times their initial body mass, leads to rapid activation of high output by excretory system, which eliminates 40-50 percent of the fluid mass. Secretion of ions and water is stimulated 1000-fold by serotonin and diuretic hormone. These hormones cooperate synergistically to activate adenylate cyclase activity from MTs cells, which increase the level of intracellular cAMP. The anti-diuretic hormones have also an important role in the fluid maintenance of Rhodnius prolixus. Several hours after insect feeding occurs a reduction in urine flow, that has been thought to result from a decreased diuretic hormone release or from a novel mechanism of anti-diuresis involving insect cardioacceleratory peptide 2b (CAP2b) and cyclic GMP. In this article it is discussed how the hormone regulation of fluid transport is done in Rhodnius prolixus MTs.

Os túbulos de Malpighi (TMs) de Rhodnius prolixus são reconhecidos por serem excelentes modelos para o estudo da secreção de fluidos e íons devido a grande diurese que ocorre quando esses animais se alimentam de sangue. O inseto, após alimentação, pode aumentar seu peso corporal inicial em até 10-12 vezes, o que leva a rápida ativação do sistema excretor, que elimina 40-50 por cento do fluido corporal. A secreção de íons e água é estimulada 1000 vezes pela serotonina e pelos hormônios diuréticos. Esses hormônios agem sinergicamente ativando a adenil ciclase das células dos TMs, aumentando os níveis intracelulares de AMPc. Os hormônios anti-diuréticos também têm um importante papel na manutenção dos fluídos corporais do Rhodnius prolixus. Várias horas após a alimentação do inseto ocorre uma redução do fluxo urinário, o que foi sugerido ser decorrente da diminuição da liberação dos hormônios diuréticos ou da anti-diurese envolvendo o peptídeo cardioaceleratório 2b (CAP2b) e o GMPc. Neste artigo é discutida a regulação hormonal do transporte de fluido nos MTs de Rhodnius prolixus.

Cell markers for ecotoxicological studies in target organs of bees

Bees are important pollinators that, because of extensive deforestation of their natural habitats, now forage widely in agricultural areas. This interaction with human agricultural activity has led to a reduction in the number of bee species because of contact with widely used pesticides. However, little is known about the adverse effects that exposure to such agents has on bee tissues and organs. In this review, we discuss the morphological alterations induced by environmental contaminants in the midgut and Malpighian tubules of bees; these two organs are involved in the absorption and excretion of toxic compounds, respectively. We also discuss the role of heat shock proteins, also known as stress proteins, in the cellular response to chemical compounds, and the importance of cell death as an indicator of the toxicity of these compounds. The analysis of these two cellular markers may be useful for monitoring bees that forage in agricultural areas.

Brochosomes-for-eggs of the Proconiini ( Hemiptera: Cicadellidae, Cicadellinae ) species associated with orchards of Citrus sinensis ( L. ) Osbeck in Rio Grande do Sul, Brazil

Brocossomos são corpos ultramicroscópicos, reticulados, produzidos pelos tubos de Malpighi das cigarrinhas. São geralmente esféricos (brocossomos de tegumento) ou alongados (brocossomos de ovos). Neste estudo, as espécies de Proconiini foram identificadas de acordo com seus brocossomos de ovos. As características e uma chave para a identificação dos brocossomos também foram discutidas. Os espécimes foram coletados em nove pomares de Citrus sinensis ('Valencia'), com o uso da armadilha adesiva de cor amarela. Os pomares estão localizados em sete municípios do Rio Grande do Sul: Tenente Portela, Ijuí, Jaguari, Harmonia, Taquari, Montenegro e Pelotas. Os dados foram coletados de outubro de 1999 a dezembro de 2000 e de outubro de 2001 a março de 2002. Foram identificadas sete espécies capazes de produzir brocossomos de ovos: Acrogonia citrina Marucci & Cavichioli, Homalodisca ignorata Melichar, Molomea consolida Shröder, Molomea lineiceps Young, Molomea magna (Walker), Oncometopia facialis (Signoret), e Oncometopia fusca Melichar. Este estudo demonstra que a diversidade de brocossomos de ovos pode ser uma ferramenta útil para a identificação de espécies de Proconiini.

Estudio de los mecanismos de transporte iónico involucrados en la secreción en túbulos de malpighi de chipo (Rhodnius prolixus) / Study of the mechanism of ionic transport involved on the secretion of malpighian tubes of chipos (Rhodnius prolixus)

Se midió la tasa de secreción y los cambios en la concentración interna de sodio y protones de los túbulos demalpighi (UMT) en Rhodnius prolixus bajo estimulación con 5-OH-Triptamina. Para medir secreción utilizamos un sistema de doble perfusión para tener acceso por separado a las membranas basolateral y/o apical de la célula. Los flujos iónicos se midieron por microfluorometría. Se aplicaron hasta trece agentes farmacológicos: Ouabaina, Bafilomicina A1, Furosamida, Bumetanida, DIOA, Probenecina, SITS, Acetazolamida, Amilorida, DPC, BaCl2, pCMBS y DTT. Estos agentes son bloqueadores conocidos de diferentes funciones de transporte de iones, conocidos como ATPasas, co- y/o contratransportadores asi como de canales iónicos y de agua. La asunción básica es que los cambios en los movimientos de agua reflejan los cambios en los mecanismos del transporte de iones que se localizan de la siguiente manera: (i) en la membrana basolateral de la célula, los principales son un cotransportador de Na+-K+-2Cl– y el intercambiador de Cl--HCO- 3; la ATPasa de Na+- K+ y la ATPasa de Na+ y los canales de Cl- y de agua (Rp-MIP) son de importancia intermedia, mientras que los canales de K+ son menos importantes: (ii) en la membrana celular apical, la mayor importancia la tiene un cotransportador de K+-Cl-, que ha sido localizado por primera vez, una ATPasa de H+ tipo V, y un intercambiador de Na+-H+; el intercambiador urato-anión y los canales de K+ son importantes mientras que los canales de Clno lo son. En este trabajo presentamos un modelo actualizado que explica la secreción del UMT.

We have measured fluid secretion rate and intracelular concentration of sodium and protons, in Rhodnius prolixusUMT stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. The ionic fluxes has been measured using microfluorimetry. 13 pharmacological agents were applied: Ouabain, Bafilomycin A1, Furosemide, Bumetanide, DIOA, Probenecid, SITS, Acetazolamide, Amiloride, DPC, BaCl2, pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter- transporters and ionic and water channels. The basic assumption is that water movement changes reflect changes on ion transport mechanisms which we localize as follows: (i) At the basolateral cell membrane: a Na+ - K+ - 2Cl- cotransport; a Cl- - HCO3 - exchange; the Na+ - K+ - ATPase, Cl- channels and Rp-MIP water channels are fundamental; K+ channels play a lesser role. (ii) At the apical cell membrane, a K+ - Cl- cotransport that is located for the first time; a V - H+ - ATPase; an Na+– H+ exchange; an urate - anion exchange and K+ channels are important, while Cl- channels are not. The present experiments allow us to build a tentative model for the function of the UMT cell, which includes a paracellular pathway for fluid flow. In this work we propose a new actualized model to explain the UMT secretion.

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.

In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae)

Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae)

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20ºC) and old (20 to 42 h, 28ºC) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed

A technique for preparing polytene chromosomes from Aedes aegypti (Diptera, Culicinae)

Polytene chromosome preparations were obtained from larval, pupal and adult female Malpighian tubules of Aedes aegypti. The Malpighian tubules of the pupae (0-4 h old) from larvae reared at 20ºC provided the best cytogenetic analysis. The interaction of nucleic acids and proteins that influence the spreading of the chromosomes could be reduced with the preparation technique of the sheets submitted to a stronger treatment starting with the hypotony of tissue and successive bathings with acetic acid. A simple technique should facilitate molecular cytogenetics used in the location of resistance and vector competence genes

Encuentro con Rhodnius prolixus y su sistema excretor / Encounter with Rhodnius prolixus and its excretory system

El Rhodnius prolixus (chipo) es uno de los vectores del Trypanosoma cruzi ya que sus deyecciones pueden contener las formas metacíclicas del parásito, transmitiendo la enfermedad de Chagas. Más allá de su relación con la enfermedad, resulta de interés reconocer y admirar la sencillez y eficacia de su sistema excretor que le permite eliminar, en pocas horas, gran parte del agua y las sales contenidas en la sangre succionada. En este artículo se relata el encuentro que tuvo Charles Darwin con las "benchugas" en 1835 y se describe cómo el insecto puede aumentar 10 veces su peso después de ser colocado ante un alimentador con sangre humana heparinizada a 37 ºC y cómo la sangre puede ser reemplazada por Ringer + ATP. La excreción está regulada por la acción de las hormonas diurética y serotonina, actuando en forma sinérgica. Estas co-hormonas tiene como segundo mensajero al AMPc y es posible encontrar al péptido en la orina y heces del R. prolixus asi como en el fluido producido por los túbulos de Malpighi. El AMPc podría ser un factor inductor de la metaciclogénesis del T. cruzi, completando el ciclo del parásito. Por su parte, la serotonina también actúa ablandando la unión de las placas cituculares del insecto, permitiendo la distensión de su abdomen durante la ingestión de sangre o Ringer y su acción puede ser inhibida por antagonista. En este artículo se describe un método para demostrar en el insecto entero la acción de la serotonina y los antagonistas sobre los túbulos de Mailpighi. El R. prolixus inyecta, para facilitar su ingesta de sangre, dadores de óxido nitrico, el que actua como vasodilatador y antiagregante plaquetario. El R. prolixus es, estonces, un excelente modelo que permite, mediante un simple ejercicio de fisiología comparada, comprender mecanismos fisiológicos del hombre y otros animales más evolucionados

Estudo morfo-fisiológico dos túbulos de Malpighi de Rhodnius prolixus stal 1859 (Hemiptera, Reduviidae, Triatominae) / Morpho-physiological study of malpighian tubules of Rhodnius prolixus stal 1859 (Hemiptera, Reduviidae, Triatominae)

Foram analisadas modificações nos túbulos de Malpighi de Rhodnius prolixus durante o ciclo alimentar do inseto. As regiões proximais e distais destes túbulos, possuem epitélio simples, envoltos por uma lâmina basal. O citoplasma é homogêneo na região proximal, existindo na distal, numerosos vacúolos, grânulos e concreções de minerais amorfos... A região proximal apresenta interdigitações basais, de aspecto vacuolizado e na região distal estas têm aspecto paralelo. O glicogênio evidenciado nos túbulos de Malpighi é possivelmente a principal fonte de reserva de energia nestas células. A partir de uma hora após a alimentação, a região proximal apresenta uma gradual diminuição do volume do epitélio, com intensa vacuolização na região basal e desorganização parcial das microvilosidades. Com 72h o volume é grande e as mitocôndrias encontram-se espalhadas pelo epitélio. A região distal apresenta concreções de minerais amorfos com aspecto de lamelas concêntricas. Após 24h observa-se muitos vacúolos de conteúdo não-eletrondenso. Os túbulos, quando submetidos a um choque hiperosmótico, apresentam uma desorganização parcial das microvilosidades na região proximal, sem alteração da vacuolização basal. Na região distal, as concreções são expelidas para o lúmen juntamente com porções de membrana, sem quebra da integridade do epitélio, mantendo o padrão em paralelo das interdigitações basais, sugerindo que os íons presentes seriam utilizados na regulação de volume. A ocorrência de uma variação na expressão da Na++K+-ATPase indica que esta enzima participa ativamente da regulação do volume. O mesmo ocorre com VH+-ATPase, cuja intensa atividade expressa com uma hora após o repasto, coincide com o pico de presença de cálcio e fósforo nas concreções de minerais amorfos, com a detecção da Ca++-ATPase por citoquímica ultraestrutural e pela alta atividade da Na++K+-ATPase neste período. O citoesqueleto evidenciou uma variação na expressão da tubulina nos diferentes tempos após a alimentação, diferente dos filamentos de actina, que mantiveram um padrão constante. Estes dados reforçam a importância da alimentação nas modificações morfológicas, na regulação do volume celular e na expressão das ATPases da membrana luminal e baso-lateral nas células dos túbulos de Malpighi de R. prolixus.

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

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

Sodium pumps in the Malpighian tubule of Rhodnius sp

Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+)ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+)ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+)ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+)ATPase activity. Taken together these data indicate that (Na+ + K+)ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.

Prevalência da microbiota no trato digestivo de fêmeas de Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera, Psychodidae) provenientes do campo / Prevalence of the microbiota in the digestive tract of wild-caught females of Lutzomyia longipalpis (Lutz & Neiva) (Diptera, Psychodidae)

No presente trabalho foram dissecados o trato digestivo de 245 fêmeas de Lutzomyia longipalpis originários da Gruta da Lapinha, Município de Lagoa Santa, MG, formando 7 grupos de 35 flebotomíneos. Das 8 espécies de bactérias isoladas houve uma predominância de bactérias Gram negativas (BGN) pertencentes ao grupo de näo fermentadoras de açúcar das seguintes espécies: Acinetobacter lowffii, Stenotrophomonas maltophhilia, Pseudomonas putida e Flavimonas orizihabitans. No grupo das fermentadoras tivemos: Enterobacter cloacae e Klebsiella ozaenae. No grupo dos Gram positivos foram identificados Bacillus thuringiensis e Staphylococcus spp

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.