La cytometrie en image a balayage laser (CIBL) permet de localiser et de quantifier la fluorescen... more La cytometrie en image a balayage laser (CIBL) permet de localiser et de quantifier la fluorescence au niveau tissulaire. Des observations preliminaires ont ete realisees sur des sections histologiques de larves de lepidopteres afin de mettre en evidence, apres immunomarquage en fluorescence, la distribution des sites d'accrochage des toxines de B. thuringiensis. Les premiers resultats montrent que, si le marquage visualise (et quantifie) se trouve bien au niveau des microvillosites, il n'est pas continu. Il serait preferentiellement localise dans des zones precises de l'intestin moyen de l'insecte.
Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained fr... more Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni...
L'etude du systeme phenoloxydase de l'orthoptere l. Migratoria a ete abordee. Nous avons ... more L'etude du systeme phenoloxydase de l'orthoptere l. Migratoria a ete abordee. Nous avons etudie la repartition du systeme d'activation de la prophenoloxydase dans le plasma et les hemocytes et montre son mode d'action in vitro. Contrairement au serum qui a un effet opsonisant, le plasma inhibe l'activation de la prophenoloxydase. Cet effet depresseur est du entre autre a un inhibiteur de protease que nous avons isole du plasma. Le systeme phenoloxydase intervient dans la reconnaissance des corps etrangers qui lui fournissent les signaux permettant le declenchement de la cascade de reactions conduisant a l'activation des phenoloxydases. L'etude d'une lectine qui agglutine les hematies de lapin et specifique du p-nitrophenyl d galactoside a egalement ete abordee. La purification et l'analyse biochimique ont montre qu'il s'agit d'une proteine acide de haut poids moleculaire. C'est un polymere (650 kd) dont la sous-unite a une masse molaire de 80 kd. Cette lectine se trouve dans le plasma ainsi que sur la membrane plasmique des hemocytes. Elle joue un role dans la reconnaissance immunitaire des corps etrangers qui presentent son (ses) site(s) recepteur(s)
Since the pioneer work of Swammerdam (1737), who described the haemocytes of Pediculus humanus, t... more Since the pioneer work of Swammerdam (1737), who described the haemocytes of Pediculus humanus, the blood cells of insects have been the subject of numerous investigations. Until recently, haemocyte classifications were mainly based on the morphology and staining capacities of these cells as observed under light microscopy (Wigglesworth, 1959). Several papers were published and discrepancies in haemocyte classification are obvious. For example, the same haemocyte type may be labelled differently in various insect species or even in the same species, depending on the author of the investigations. Similarly, the same name has been used to identify two (or more) different types of blood cells. This situation gave rise to much confusion among insect haematologists regarding the identification of haemocyte types and considerable disagreement as to the number of blood cell types present in various insect species.
The haemagglutinating activity in the haemolymph of Locusfa migraroria fifth-instar larvae was ex... more The haemagglutinating activity in the haemolymph of Locusfa migraroria fifth-instar larvae was examined using several vertebrate erythrocytes. No agglutination was observed using red blood cells from chicken, sheep, rat, guinea pig or human A, B, 0. The highest titres were observed with rabbit erythrocytes (up to 2'*). When tested with one kind of red blood cell, the agglutinin titres were similar in the plasma and in the serum of locusts. The agglutination was heat labile (65°C for 30 min). Its titre showed a natural development in the course of the fifth larval instar. Inhibition studies with a range of carbohydrates revealed a high specificity of the agglutinin molecule for the D-galactosides with a I4 linkage. In vitro and in vioo experiments were conducted comparing the rabbit red blood cells which are strongly agglutinated, with the sheep red blood cells which are not. After injection, the clearance rate of the rabbit cells was much higher than that of the sheep cells. In vitro, the rabbit red blood cells readily attached to haemocytes and "rosette" formations were numerous whereas the sheep cells only weakly adhered to the insect blood cells and never exhibited "rosette" formations. The haemagglutinin demonstrated in the haemolymph of L. migraroriu acts as opsonin for the particles which possess receptors for this molecule.
International Journal of Tropical Insect Science, 1997
RésuméL’hémolymphe des criquets migrateurs, Schistocerca gregaria et Locusta migratoria contient ... more RésuméL’hémolymphe des criquets migrateurs, Schistocerca gregaria et Locusta migratoria contient des facteurs humoraux, la lectine et le système phénoloxydase. Ces facteurs ont été mis en évidence par des tests in vitro. La lectine présente dans le plasma agglutine les hématies de lapin et les protozoaires Malamoeba locustae, Leishmania major et Trypanosoma brucei. Sa purification et son analyse par électrophorèse en conditions dénaturantes et non dénaturantes ont révélé qu’il s’agit d’une protéine de 650 kD composée de sous-unités de 80 kD. Le système phénoloxydase a été également étudié. Il peut être activé in vitro par des substances d’origine microbienne (LPS, laminarine, suspension de kystes de M. locustae), par la trypsine ou le méthanol. Les inhibiteurs de ces deux facteurs humoraux ont été étudiés. Dans le cas de la lectine, il s’agit du sucre inhibiteur, le nitrophényl ± d-galactopyranoside et l’antisérum antilectine. Des inhibiteurs du système phénoloxydase ont été identifiés dans le plasma. Les perspectives d’utilisation de ces inhibiteurs dans la lutte biologique sont discutées. En effet, il serait intéressant d’étudier les possibilités de leur incorporation dans la formulation des biopesticides; ils pourraient ainsi exercer une action dépressive sur le système immunitaire de l’insecte tout en amplifiant l’action des biopesticides.AbstractHaemolymph from the locusts Schistocerca gregaria and Locusta migratoria contains humoral factors such as lectin and phenoloxidase system. In vitro studies demonstrated that phenoloxidase system can be activated by LPS, laminarin, Malamoeba locustae cysts, trypsin and methanol. Haemolymph showed agglutination activity for mammalian erythrocytes and protozoa (M. locustae, Leishmania major and Trypanosoma brucei). A purified lectin was isolated from haemolymph by affinity chromatography. Analysis by SDS-PAGE gave a single band at 80 kD while on native PAGE, it gave a single band at 650 kD. Inhibitors of the two humoral factors were investigated. Lectin activity was inhibited by nitrophenyl ± d-galactopyranoside in both cases and by lectin antiserum. Inhibitors of the phenoloxidase system were found in plasma. Prospects for use of such inhibitors are discussed. They could be incorporated in biopesticide formulations in such a way that they could depress locust immune reaction and stimulate biocide activity.
An agglutinin from plasma of the locust Locusta migratoria was purified to apparent homogeneity b... more An agglutinin from plasma of the locust Locusta migratoria was purified to apparent homogeneity by one step affinity chromatography. Two kinds of affinity columns were used which gave the same results concerning the degree of purification. We named this agglutinin 'migratorin'. Its subunit Mr is 80 kDa with no disulfide bounds. Measurements of M, of the native protein by Ferguson plots gave M, c. 650 kDa, which means that the agglutinin is probably a polymer of 8 subunits. Its pI is 5.8. These M, and pI are similar to those of several insect lectins. The purified agglutinin is heat resistant. Agglutination of RRBC required divalent cations. Some sugars were inhibitors of agglutination by crude plasma or by migratorin. Agglutination was also inhibited in both crude plasma and migratorin by antibodies raised against purified migratorin. These data indicate that agglutination observed in locust hemolymph was due to the protein we have purified. Insect immunity Agglutinin Lectin Non-self recognition Isoelectrofocusing Affinity chromatography
Abstract The hemocyte types of Culex pipiens and Aedes aegypti larvae are described. Plasmatocyte... more Abstract The hemocyte types of Culex pipiens and Aedes aegypti larvae are described. Plasmatocytes and oenocytoids are present and are involved in phagocytosis and melanin production, respectively. Comparisons are made with the findings of previous authors and together with modifications in the hemogram following parasitization by the nematode, Neoplecta carpocapsae , indicate that the hemocytes are also involved in encapsulation reactions.
In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amount... more In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amounts relative to the haemocytes. This phenoloxidase activity cannot be induced by either heating or freezing and thawing and it is lost by heating at 70°C for 30 rain. Both iipopolysaccharides and laminarin can elicit the prophenoloxidase activating system. These elicitors of prophenoloxidase activation are active in haemocyte lysate and in serum but never induce any phenoloxidase activity in plasma. In haemocyte lysate, the activating system is not heat resistant, and heating at 56°C for 30 rain prior to incubation with laminarin or lipopolysaccharide precludes any phenoloxidase activity. Plasma contains a strong inhibitor of the prophenoloxidase activating system but serum does not. This inhibitor does not affect the phenoloxidase enzyme itself. The possible role of the activating system in immune recognition and the strategies evolved by parasites or pathogens to escape being recognized by their host are discussed.
La cytometrie en image a balayage laser (CIBL) permet de localiser et de quantifier la fluorescen... more La cytometrie en image a balayage laser (CIBL) permet de localiser et de quantifier la fluorescence au niveau tissulaire. Des observations preliminaires ont ete realisees sur des sections histologiques de larves de lepidopteres afin de mettre en evidence, apres immunomarquage en fluorescence, la distribution des sites d'accrochage des toxines de B. thuringiensis. Les premiers resultats montrent que, si le marquage visualise (et quantifie) se trouve bien au niveau des microvillosites, il n'est pas continu. Il serait preferentiellement localise dans des zones precises de l'intestin moyen de l'insecte.
Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained fr... more Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni...
L'etude du systeme phenoloxydase de l'orthoptere l. Migratoria a ete abordee. Nous avons ... more L'etude du systeme phenoloxydase de l'orthoptere l. Migratoria a ete abordee. Nous avons etudie la repartition du systeme d'activation de la prophenoloxydase dans le plasma et les hemocytes et montre son mode d'action in vitro. Contrairement au serum qui a un effet opsonisant, le plasma inhibe l'activation de la prophenoloxydase. Cet effet depresseur est du entre autre a un inhibiteur de protease que nous avons isole du plasma. Le systeme phenoloxydase intervient dans la reconnaissance des corps etrangers qui lui fournissent les signaux permettant le declenchement de la cascade de reactions conduisant a l'activation des phenoloxydases. L'etude d'une lectine qui agglutine les hematies de lapin et specifique du p-nitrophenyl d galactoside a egalement ete abordee. La purification et l'analyse biochimique ont montre qu'il s'agit d'une proteine acide de haut poids moleculaire. C'est un polymere (650 kd) dont la sous-unite a une masse molaire de 80 kd. Cette lectine se trouve dans le plasma ainsi que sur la membrane plasmique des hemocytes. Elle joue un role dans la reconnaissance immunitaire des corps etrangers qui presentent son (ses) site(s) recepteur(s)
Since the pioneer work of Swammerdam (1737), who described the haemocytes of Pediculus humanus, t... more Since the pioneer work of Swammerdam (1737), who described the haemocytes of Pediculus humanus, the blood cells of insects have been the subject of numerous investigations. Until recently, haemocyte classifications were mainly based on the morphology and staining capacities of these cells as observed under light microscopy (Wigglesworth, 1959). Several papers were published and discrepancies in haemocyte classification are obvious. For example, the same haemocyte type may be labelled differently in various insect species or even in the same species, depending on the author of the investigations. Similarly, the same name has been used to identify two (or more) different types of blood cells. This situation gave rise to much confusion among insect haematologists regarding the identification of haemocyte types and considerable disagreement as to the number of blood cell types present in various insect species.
The haemagglutinating activity in the haemolymph of Locusfa migraroria fifth-instar larvae was ex... more The haemagglutinating activity in the haemolymph of Locusfa migraroria fifth-instar larvae was examined using several vertebrate erythrocytes. No agglutination was observed using red blood cells from chicken, sheep, rat, guinea pig or human A, B, 0. The highest titres were observed with rabbit erythrocytes (up to 2'*). When tested with one kind of red blood cell, the agglutinin titres were similar in the plasma and in the serum of locusts. The agglutination was heat labile (65°C for 30 min). Its titre showed a natural development in the course of the fifth larval instar. Inhibition studies with a range of carbohydrates revealed a high specificity of the agglutinin molecule for the D-galactosides with a I4 linkage. In vitro and in vioo experiments were conducted comparing the rabbit red blood cells which are strongly agglutinated, with the sheep red blood cells which are not. After injection, the clearance rate of the rabbit cells was much higher than that of the sheep cells. In vitro, the rabbit red blood cells readily attached to haemocytes and "rosette" formations were numerous whereas the sheep cells only weakly adhered to the insect blood cells and never exhibited "rosette" formations. The haemagglutinin demonstrated in the haemolymph of L. migraroriu acts as opsonin for the particles which possess receptors for this molecule.
International Journal of Tropical Insect Science, 1997
RésuméL’hémolymphe des criquets migrateurs, Schistocerca gregaria et Locusta migratoria contient ... more RésuméL’hémolymphe des criquets migrateurs, Schistocerca gregaria et Locusta migratoria contient des facteurs humoraux, la lectine et le système phénoloxydase. Ces facteurs ont été mis en évidence par des tests in vitro. La lectine présente dans le plasma agglutine les hématies de lapin et les protozoaires Malamoeba locustae, Leishmania major et Trypanosoma brucei. Sa purification et son analyse par électrophorèse en conditions dénaturantes et non dénaturantes ont révélé qu’il s’agit d’une protéine de 650 kD composée de sous-unités de 80 kD. Le système phénoloxydase a été également étudié. Il peut être activé in vitro par des substances d’origine microbienne (LPS, laminarine, suspension de kystes de M. locustae), par la trypsine ou le méthanol. Les inhibiteurs de ces deux facteurs humoraux ont été étudiés. Dans le cas de la lectine, il s’agit du sucre inhibiteur, le nitrophényl ± d-galactopyranoside et l’antisérum antilectine. Des inhibiteurs du système phénoloxydase ont été identifiés dans le plasma. Les perspectives d’utilisation de ces inhibiteurs dans la lutte biologique sont discutées. En effet, il serait intéressant d’étudier les possibilités de leur incorporation dans la formulation des biopesticides; ils pourraient ainsi exercer une action dépressive sur le système immunitaire de l’insecte tout en amplifiant l’action des biopesticides.AbstractHaemolymph from the locusts Schistocerca gregaria and Locusta migratoria contains humoral factors such as lectin and phenoloxidase system. In vitro studies demonstrated that phenoloxidase system can be activated by LPS, laminarin, Malamoeba locustae cysts, trypsin and methanol. Haemolymph showed agglutination activity for mammalian erythrocytes and protozoa (M. locustae, Leishmania major and Trypanosoma brucei). A purified lectin was isolated from haemolymph by affinity chromatography. Analysis by SDS-PAGE gave a single band at 80 kD while on native PAGE, it gave a single band at 650 kD. Inhibitors of the two humoral factors were investigated. Lectin activity was inhibited by nitrophenyl ± d-galactopyranoside in both cases and by lectin antiserum. Inhibitors of the phenoloxidase system were found in plasma. Prospects for use of such inhibitors are discussed. They could be incorporated in biopesticide formulations in such a way that they could depress locust immune reaction and stimulate biocide activity.
An agglutinin from plasma of the locust Locusta migratoria was purified to apparent homogeneity b... more An agglutinin from plasma of the locust Locusta migratoria was purified to apparent homogeneity by one step affinity chromatography. Two kinds of affinity columns were used which gave the same results concerning the degree of purification. We named this agglutinin 'migratorin'. Its subunit Mr is 80 kDa with no disulfide bounds. Measurements of M, of the native protein by Ferguson plots gave M, c. 650 kDa, which means that the agglutinin is probably a polymer of 8 subunits. Its pI is 5.8. These M, and pI are similar to those of several insect lectins. The purified agglutinin is heat resistant. Agglutination of RRBC required divalent cations. Some sugars were inhibitors of agglutination by crude plasma or by migratorin. Agglutination was also inhibited in both crude plasma and migratorin by antibodies raised against purified migratorin. These data indicate that agglutination observed in locust hemolymph was due to the protein we have purified. Insect immunity Agglutinin Lectin Non-self recognition Isoelectrofocusing Affinity chromatography
Abstract The hemocyte types of Culex pipiens and Aedes aegypti larvae are described. Plasmatocyte... more Abstract The hemocyte types of Culex pipiens and Aedes aegypti larvae are described. Plasmatocytes and oenocytoids are present and are involved in phagocytosis and melanin production, respectively. Comparisons are made with the findings of previous authors and together with modifications in the hemogram following parasitization by the nematode, Neoplecta carpocapsae , indicate that the hemocytes are also involved in encapsulation reactions.
In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amount... more In Locusta migratoria, prophenoloxidase is present in the plasma and serum, but in reduced amounts relative to the haemocytes. This phenoloxidase activity cannot be induced by either heating or freezing and thawing and it is lost by heating at 70°C for 30 rain. Both iipopolysaccharides and laminarin can elicit the prophenoloxidase activating system. These elicitors of prophenoloxidase activation are active in haemocyte lysate and in serum but never induce any phenoloxidase activity in plasma. In haemocyte lysate, the activating system is not heat resistant, and heating at 56°C for 30 rain prior to incubation with laminarin or lipopolysaccharide precludes any phenoloxidase activity. Plasma contains a strong inhibitor of the prophenoloxidase activating system but serum does not. This inhibitor does not affect the phenoloxidase enzyme itself. The possible role of the activating system in immune recognition and the strategies evolved by parasites or pathogens to escape being recognized by their host are discussed.
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