RESUMO
Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three ß-glucosidases, one ß-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none ß-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single ß-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two ß-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of ß-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided.
Assuntos
Digestão/fisiologia , Periplaneta/fisiologia , Aminopeptidases/genética , Aminopeptidases/fisiologia , Animais , Sequência de Bases , Quitinases/genética , Quitinases/fisiologia , Quimotripsina/genética , Quimotripsina/fisiologia , Trato Gastrointestinal/anatomia & histologia , Trato Gastrointestinal/diagnóstico por imagem , Glucosidases/genética , Glucosidases/fisiologia , Microscopia Eletrônica , Dados de Sequência Molecular , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/fisiologia , Periplaneta/anatomia & histologia , Periplaneta/enzimologia , Periplaneta/genética , Reação em Cadeia da Polimerase , Transcriptoma/genética , Tripsina/genética , Tripsina/fisiologia , Ultrassonografia , beta-Galactosidase/genética , beta-Galactosidase/fisiologia , beta-Glucosidase/genética , beta-Glucosidase/fisiologiaRESUMO
Austin (AT) and its derivatives (dehydroaustin (DAT) and acetoxydehydroaustin (ADAT)) produced by Penicillium brasilianum MG-11 exhibit toxicity to insects, yet their targets are unknown. Here, we used whole-cell patch-clamp electrophysiology to investigate the action of AT family compounds on cockroach acetylcholine (ACh), γ-aminobutyric acid (GABA) and l-glutamate receptors expressed in the American cockroach (Periplaneta americana) neuron. U-tube application of AT or its derivatives did not induce any current amplitudes, suggesting that they did not act as agonist of these three receptors. In the second step of experiments, they were bath-applied for 1min before co-application with the corresponding ligand. We found that AT and its derivatives had no effect on GABA and l-glutamate-induced currents, whereas they significantly reduced ACh- and epibatidine-induced currents, showing that these compounds acted as selective antagonists of nicotinic acetylcholine receptors (nAChRs) expressed in the cockroach neuron. Of the compounds, DAT showed the highest blocking potency for nAChRs, differentially attenuating the peak and slowly desensitizing current amplitude of ACh-induced responses with pIC(50) (=-logIC(50) (M)) values of 6.11 and 5.91, respectively. DAT reduced the maximum normalized response to ACh without a significant shift in EC(50), suggesting that the blocking action is not competitive with ACh.
Assuntos
Antagonistas Nicotínicos/toxicidade , Penicillium , Periplaneta/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Terpenos/toxicidade , Animais , Baratas , Relação Dose-Resposta a Droga , Masculino , Antagonistas Nicotínicos/isolamento & purificação , Penicillium/isolamento & purificação , Periplaneta/fisiologia , Receptores Nicotínicos/fisiologia , Terpenos/isolamento & purificaçãoRESUMO
In the larval cockroach (Periplaneta americana), knockout of Engrailed (En) in the medial sensory neurons of the cercal sensory system changes their axonal arborization and synaptic specificity. Immunocytochemistry has been used to investigate whether the co-repressor Groucho (Gro; vertebrate homolog: TLE) and the co-factor Extradenticle (Exd; vertebrate homolog: Pbx) are expressed in the cercal system. Gro/TLE is expressed ubiquitously in cell nuclei in the embryo, except for the distal pleuropodia. Gro is expressed in all nuclei of the thoracic and abdominal central nervous system (CNS) of first instar larva, although some neurons express less Gro than others. Cercal sensory neurons express Gro protein, which might therefore act as a co-repressor with En. Exd/Pbx is expressed in the proximal portion of all segmental appendages in the embryo, with the exception of the cerci. In the first instar CNS, Exd protein is expressed in subsets of neurons (including dorsal unpaired medial neurons) in the thoracic ganglia, in the first two abdominal ganglia, and in neuromeres A8-A11 of the terminal ganglion. Exd is absent from the cerci. Because Ultrabithorax/Abdominal-A (Ubx/Abd-A) can substitute for Exd as En co-factors in Drosophila, Ubx/Abd-A immunoreactivity has also been investigated. Ubx/Abd-A immunostaining is present in abdominal segments of the embryo and first instar CNS as far caudal as A7 and faintly in the T3 segment. However, Ubx/Abd-A is absent in the cerci and their neurons. Thus, in contrast to its role in Drosophila segmentation, En does not require the co-factors Exd or Ubx/Abd-A in order to control the synaptic specificity of cockroach sensory neurons.
Assuntos
Sistema Nervoso Central/metabolismo , Gânglios dos Invertebrados/metabolismo , Gânglios Sensitivos/metabolismo , Proteínas de Homeodomínio/metabolismo , Periplaneta/fisiologia , Proteínas Repressoras/metabolismo , Animais , Feminino , Estágios do Ciclo de Vida/fisiologia , Fatores de TranscriçãoRESUMO
In this work, phosphatase activity was characterized in the ovary and the haemolymph of Periplaneta americana. The optimum pH for these activities was 4.0, and a temperature of 44 degrees C was ideal for the maximal enzyme activity. The phosphatase activities were inhibited by NaF, sodium tartrate, Pi, sodium orthovanadate, and ammonium molybdate. The ovarian phosphatase activity at pH 4.0 was almost exclusive against phosphotyrosine, with little or no effect on the residues of phosphoserine or phosphothreonine. These results indicate that this phosphatase activity is due to the presence of an acid tyrosine phosphatase. The phosphatase activities of acid extracts from P. americana ovaries (OEX) and an acid extract from P. americana haemolymph (HEX) were analyzed in non-denaturant gel electrophoresis using an analog substrate beta-naphtyl phosphate. The gel revealed two bands with phosphatase activity in the ovary and one band in the haemolymph; these bands were excised and submitted to a 10% SDS-PAGE showing a single 70-kDa polypeptide in both samples. Histochemistry of the ovary with alpha-naphtyl phosphate for localization of acid phosphatase activity showed mainly labeling associated to the oocyte peripheral vesicles, basal lamina, and between follicle cells. Electron microscopy analysis showed that acid phosphatase was localized in small peripheral vesicles in the oocyte, but not inside yolk granules. The possible role of this phosphatase during oogenesis and embryogenesis is also discussed in this article.
Assuntos
Oogênese/fisiologia , Periplaneta/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Animais , Feminino , Histocitoquímica , Microscopia Eletrônica de Transmissão , Ovário/enzimologia , Ovário/fisiologia , Ovário/ultraestrutura , Periplaneta/enzimologia , Periplaneta/ultraestrutura , Proteínas Tirosina Fosfatases/metabolismoRESUMO
The synaptic connection between the filiform hair sensory neurons (FHSNs) and the giant interneurons (GIs) in the terminal abdominal ganglion of the first-instar cockroach Periplaneta americana has been used as a model system for investigating the mechanism of synaptic specificity. Electrophysiological and electron microscopical techniques were used to determine the normal patterns of connectivity between FHSNs and GIs. It was found that, even during synaptogenesis, no incorrect synapses were formed between the sensory axons and the interneuronal dendrites. Studies of a mutant in which there is a supernumerary filiform hair have given an insight into the role of positional information in determining the shape of a sensory neuron, and its connectivity with the GIs. There is electrophysiological evidence for competitive interactions between the supernumerary sensory axon and the original.
Assuntos
Baratas/fisiologia , Metamorfose Biológica , Fenômenos Fisiológicos do Sistema Nervoso , Periplaneta/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Animais , Axônios/fisiologia , Dendritos/fisiologia , Gânglios/fisiologia , Interneurônios/fisiologia , Mutação , Neurônios/fisiologia , Orientação/fisiologia , Células Receptoras Sensoriais/fisiologiaRESUMO
Las conecciones sinápticas entre las neuronas sensoriales de los pelos filiformes (FHSNs) y las interneuronas gigantes (GIs) en el ganglio terminal abdominal del primer estadió ninfal de la cucaracha Periplaneta americana han sido utilizado como sistema modelo para investigar los mecanismos de especificidad en la formación de sinapsis. Técnicas de electrofisiología y microscopía electrónica fueron usadas con el fin de establecer los circuitos normales de conección entre FHSNs y GIs. Se encontró que durante el período de "sinaptogénesis" no hubo equivocación al formarse las sinápsis entre los axones sensoriales y las dendritas de las interneuronas correspondientes. Estudios con un mutante que posee un axon sensorial adicional han arrojado nueva luz sobre la función de la información posicional para determinar la forma y las conecciones de la neurona sensorial con las GIs. Ha sido obtenida evidencia electrofisiológica de interacciones competitivas entre el axon sensorial adicional y el original