Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin

BUPM95 is a Bacillusthuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22 kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62 kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65 kDa compared to the 55 and 100 kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.     

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida,Phasmatidae): A morphological,physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.     

Antibiotic treatment (Tetracycline) effect on bio-efficiency of the larvae honey bee (Apis mellifera jemenatica)

Honey bees are important for ecological health, biodiversity preservation, and crop output. Antimicrobials, like Tetracyclines, are commonly used in agriculture, medicine, and beekeeping, bees might be exposed to Tetracycline residues in the environment either directly or indirectly. This study aimed to determine the effect of antibiotic treatment (Tetracycline) effect on the Bio-efficiency of the larvae honey bee (Apis mellifera jemenatica), when larvae honeybee workers were exposed to different concentrations of it, to see how long they survived after being exposed to it and affected this antibiotic to the histological structure of the midgut. The results demonstrated that the concentration (LC₅₀ = 125.25 μg/ml) of antibiotics Tetracycline leads to kills half of the individuals. Our data indicate that the high concentrations of Tetracycline have a significant effect on the histological composition of the cells of the midgut of honeybee larvae. Antibiotic exposure can negatively impact the health of honey bees, especially Tetracycline because it is the most used antibiotic in apiculture.     

Proteomic analysis of the mosquito Aedes aegypti midgut brush border membrane vesicles

We analyzed brush border membrane vesicle proteins from isolated midguts of the mosquito Aedes aegypti, by two proteomic methods: two-dimensional gel electrophoresis (isoelectric focusing and SDS-PAGE) and a shotgun two-dimensional liquid chromatographic (LS/LS) approach based on multidimensional protein identification technology (MudPIT). We were interested in the most abundant proteins of the apical brush border midgut membrane. About 400 spots were detected on 2D gels and 39 spots were cored and identified by mass spectrometry. 86 proteins were identified by MudPIT. Three proteins, arginine kinase, putative allergen and actin are shown to be the most predominant proteins in the sample. The total number of 36 proteins detected by both methods represents the most abundant proteins in the BBMV.     

Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.)

Summary A polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin.At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared.The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.     

Etude de la chambre filtrante de Planococcus citri (Insecta,Homoptera)

Résumé Le tube digestif des cochenilles se caractérise par la présence d'un dispositif intestinal appelé chambre filtrante. Celle-ci, formée par l'accolement des deux extrêmités de l'intestin moyen, est supposée intervenir dans le transit alimentaire en éliminant par «voie raccourcie» l'eau ingérée en excès, mais ce rôle est resté jusque là hypothétique.Chez Planococcus citri, l'étude cytologique et histochimique révèle des caractères propres aux parties de l'intestin moyen participant à la réalisation de ce dispositif. Ces caractères suggèrent une fonction particulière qui peut être liée à un mouvement de l'eau. Les cellules sont de taille très réduite. Par endroits, des différences ultrastructurales s'observent entre les deux anses accolées (présence de vacuoles et d'espaces extracellulaires développés). Les cellules non modifiées de l'anse de départ présentent un caractère morphologique particulier, le système d'espaces intercellulaires et un caractère histochimique dominant, l'importante quantité de glycogène. Les cellules non modifiées de l'anse de retour présentent une structure inhabituelle, les «expansions tubulaires» associées aux microvillosités. En absence du «plasmalemma mitochondrial complex» le passage de l'eau se ferait par osmose dans deux directions: d'une part à travers les épithéliums amincis, d'autre part par le réseau d'espaces intercellulaires.

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Studies on the filter-chamber of Planococcus citri risso (Insecta, Homoptera)Histochemistry and ultrastructure

Summary The digestive tract of the coccids is characterized by the presence of a filter chamber formed by the apposition of the two extremities of the midgut. It is supposed that the filter chamber plays a role in the elimination of excess water by a short circuit but this assumption is still hypothetical.In Planococcus citri, cytological and histochemical studies have revealed structural peculiarities in those parts of the midgut which participate in the organization of the filter chamber, which suggest a particular function. The cells are highly reduced in size. In certain regions ultrastructural differences exist between the two apposed extremities of the midgut such as the presence of a vacuole and of highly developed extra-cellular spaces. These features may be related to water transport. The conventional cells of the proximal end of the midgut show the presence of a system of intercellular spaces and are very rich in glycogen. The conventional cells of the distal end of the midgut possess extracellular microtubules associated with the microvilli. In absence of a plasmalemma mitochondrial complex the transport of water could occur by osmosis across the thinned epithelium and the network of intercellular spaces.

Cet article recouvre en partie le travail d'une thèse de Docteur-Ingénieur soutenue à l'Université Paris VI en 1972.     

Recherches histophysiologiques sur le développement post-embryonnaire et le cycle annuel de Formica (Hyménoptère)

Résumé Les cellules du mésentéron des ouvrières, des reines et des mâles de Formica polyctena F. possèdent un certain nombre de particularités cytologiques dont l'évolution a été suivie au cours du développement post-embryonnaire et du cycle annuel.A l'apex des cellules de régénération les microvillosités se différencient avant l'élimination des cellules caduques larvaires ou nymphales. A partir de la nymphose une activité sécrétoire apocrine se manifeste dans la partie dorsale de l'épithélium du mésentéron, l'ensemble des cellules assurant par ailleurs la fonction absorbante de l'organe. Il existe deux sortes d'inclusions cytoplasmiques, des polysaccharides et des concrétions minérales. Les polysaccharides sont surtout abondants chez les larves et les nymphes: le glycogène, polysaccharide de réserve, est utilisé au cours de l'histogénèse; des mucopolysaccharides acides, d'origine golgienne, représentent une sécrétion muqueuse. Les sphérocristaux sont constitués de strates concentriques de phosphates et chlorures de calcium et d'une matrice de mucopolysaccharides. La cristallisation des éléments minéraux s'effectue, à partir de la nymphose seulement, dans les citernes ergastoplasmiques. Cette accumulation d'ions pourrait être en relation avec le régime alimentaire de l'insecte ou représenter une voie d'excrétion.

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Histophysiological studies on the postembryonic development and the annual cycle of Formica II. Histochemical and ultrastructural characteristics of the midgut of F. polyctena

Summary The midgut cells of workers, queens and males of the ant Formica polyctena show cytological characteristics which were studied in the course of postembryonic development and annual cycle. The microvilli of the regenerating cells appear before the elimination of the regressing larval and pupal cells. At the time of pupation, an active phase of apocrine secretion begins in the dorsal part of the midgut epithelium, while the absorptive function is carried out by all cells of the organ.Two types of cytoplasmic inclusions coexist: polysaccharides and mineral concretions. The polysaccharides are particulary abundant in larvae and pupae. Glycogen is metabolized during histogenesis; acid mucopolysaccharides, elaborated in the Golgi apparatus, represent a mucous secretion. The spherites are composed of concentric strata of calcium phosphate and chloride and a matrix of mucopolysaccharides. These minerals form in the ergastoplasmic cisternae of pupal cells only. Their accumulation could be related to the insect's diet, or it could reflect a process of excretion.

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Abréviations utilisées dans les figures B Basale anhyste - CL cellule larvaire - CR cellule de régénération - G Dictyosomes - GM Gaine musculaire - M Mitochondries - Mt Microtubules - Mv Microvillosités - R Ribosomes libres Avec la collaboration technique de Mme A. Anglo. Travail exécuté dans le cadre de la Recherche coopérative sur programme n 162 du Centre National de la Recherche Scientifique.     

The vacuolar-type ATPase from insect plasma membrane: immunocytochemical localization in insect sensilla

Summary This comparative immunocytochemical investigation provides evidence that the electrogenic potassium pump of insect sensilla is a vacuolar-type proton ATPase energizing potassium-proton antiport, as was shown recently for the electrogenic potassium pump in the larval midgut of the sphinx moth Manduca sexta. Antennal sensilla of the saturniid moth Antheraea pernyi were probed with antibodies to the midgut vacuolar-type ATPase. The monoclonal antibodies recognized their epitopes in the native and SDS-denatured state, and bound specifically to the subunit with the relative molecular mass (M_r) of 67000 (antibody 86-3) or to the subunits of M_r 28000 and 16000 (antibody 47-5). Both antibodies labelled the apical region of the auxiliary cells, as was demonstrated by immunofluorescence microscopy. Immunogold-electron microscopy localized the binding sites of the 47-5 antibody in the highly folded apical plasma membranes of the auxiliary cells. Labelling was selective and was detected in all types of examined sensilla (S. trichodea, S. styloconica, S. coeloconica). These findings are in agreement with the current view that an electrogenic potassium pump is situated in the apical plasma membrane of the auxiliary cells and that the pump is involved in driving the receptor current. They support the hypothesis that a proton-motive force generated by a vacuolar-type ATPase provides an alternative to the classical Na⁺/K⁺-ATPase to energize animal plasma membranes.