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Francesco Pennacchio - One of the best experts on this subject based on the ideXlab platform.
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Cardiochiles Nigriceps polydnavirus molecular characterization and gene expression in parasitized heliothis virescens larvae
Insect Biochemistry and Molecular Biology, 1999Co-Authors: P Varricchio, Patrizia Falabella, Rocco Sordetti, Franco Graziani, C Malva, Francesco PennacchioAbstract:Abstract Cardiochiles Nigriceps Viereck is an endophagous parasitoid of larval stages of the tobacco budworm, Heliothis virescens (F.). This hymenopteran parasitoid, belonging to the family Braconidae, is associated with a polydnavirus (CnPDV), injected at oviposition along with the egg. The infection of various tissues by CnPDV determines the suppression of the host immune system and the developmental arrest of mature host larvae. In this study, CnPDV has been characterized at the structural and molecular level. The negatively stained nucleocapsids show evident ‘end structures’ and a tail-like appendage. The CnPDV genome is typically segmented, with circular dsDNA molecules, ranging in size from 2.5 kb to more than 23 kb. The early expression pattern of CnPDV in parasitized hosts has been analysed and viral clones, genomic and cDNAs, identifying genes expressed within 48 h after parasitization have been isolated. The molecular organization of one of these genes, named CnPDV1, and its putative protein product have been determined. Significant sequence homologies with other known proteins were not detected. In situ hybridization experiments indicated that this gene is expressed in the prothoracic glands of parasitized host mature larvae. A functional analysis of CnPDV1 gene product is required to assess its possible role in the regulation of parasitoid-induced alterations of host larvae.
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prothoracic gland inactivation in heliothis virescens f lepidoptera noctuidae larvae parasitized by Cardiochiles Nigriceps viereck hymenoptera braconidae
Journal of Insect Physiology, 1998Co-Authors: Francesco Pennacchio, P Varricchio, Patrizia Falabella, Rocco Sordetti, C Malva, Bradleigh S VinsonAbstract:Abstract Heliothis virescens (F.) last instar larvae parasitized by the endophagous braconid Cardiochiles Nigriceps Viereck fail to attain the pupal stage, due to a parasitoid-induced alteration of ecdysteroid biosynthesis and metabolism. Currently available information on host prothoracic gland inactivation in this host-parasitoid system is reported here. Prothoracic glands of H. virescens mature larvae show a depressed biosynthetic activity, without undergoing gross morphological disruption. The ultrastructure of gland cells is characterized by minor parasitoid-induced changes, with the rough endoplasmic reticulum appearing more developed and electrondense than in nonparasitized controls. Eventually, the cells of prothoracic glands of parasitized host last instar larvae die but maintain their structural integrity. The inactivation of pupally committed host prothoracic glands is achieved through the disruption of the PTTH signal transduction pathway. The second messenger cAMP appears to be normally produced in response to PTTH stimulation of glands explanted from parasitized host larvae, however the downstream activation of the cAMP-dependent protein kinase does not appear to occur. In fact, a marked underphosphorylation of regulatory target proteins is observed. This underphosphorylation is associated with a significant reduction in general protein synthesis, which appears to be blocked at the translational level, to a redirection of specific protein synthesis and to a drastic suppression of ecdysteroidogenesis. These parameters appeared to be correlated in a kinetic time-course study, confirming their functional link. C. Nigriceps polydnavirus (CnPDV) plays a major role in the inactivation of pupally committed host prothoracic glands, while putative factors occurring in the host haemolymph do not seem to be of particular importance at that developmental stage. Southern blot hybridization indicates the occurrence of PKI(protein kinase inhibitor)-like genes in the C. Nigriceps genome, which, in contrast, are undetectable in H. virescens.
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regulation of heliothis virescens prothoracic glands by Cardiochiles Nigriceps polydnavirus
Archives of Insect Biochemistry and Physiology, 1998Co-Authors: Francesco Pennacchio, Patrizia Falabella, Bradleigh S VinsonAbstract:Heliothis virescens (F.) Larvae parasitized by the endophagous braconid Cardiochiles Nigriceps Viereck fail to attain the pupal stage. This developmental alteration is caused by both an inactivation of prothoracic glands of last-instar larvae and an altered ecdysone metabolism. Decrease in ecdysteroidogenesis in vitro was already evident in glands explanted from larvae that have attained the early cell formation stage (day 4 of fifth instar), 6 h after parasitoid oviposition. Ecdysteroidogenesis nearly ceased by 24 h after parasitoid oviposition. The degree of this biosynthetic depression increased as the time between parasitization and gland dissection increased. A time-course study allowed us to determine if both the degree of phosphorylation of regulatory target proteins, the rate of general protein synthesis and ecdysteroidogenesis decreased in concert over time. The results provide further evidence in support of the hypothesis that these cellular activities in prothoracic gland cells are functionally correlated in steroidogenic responses. Treatment with calyx fluid and venom of C. Nigriceps duplicates the parasitism-induced inactivation of host prothoracic glands. A 6-h conditioning in vitro of pupally committed host prothoracic glands with these parasitoid female reproductive secretions resulted in a significant depression of their ecdysteroid production. However, glands lost their sensitivity to calyx fluid and venom treatment when explanted from hosts that had already attained the cell formation stage. This was further supported by the fact that nearly all the host larvae parasitized on day 4 of fifth instar (cell formation stage) pupated, while parasitization on day 3 resulted in only 11% pupation. The coupled trioxsalen/UV irradiation treatment of C. Nigriceps calyx fluid and venom eliminated their negative effect on biosynthetic activity in vitro by host prothoracic glands. This result indirectly demonstrates that C. Nigriceps polydnavirus is the major regulating factor involved in the host prothoracic gland inactivation. Arch. Insect Biochem. Physiol. 38:1–10, 1998. © 1998 Wiley-Liss, Inc.
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biochemical and ultrastructural alterations in prothoracic glands of heliothis virescens f lepidoptera noctuidae last instar larvae parasitized by Cardiochiles Nigriceps viereck hymenoptera braconidae
Insect Biochemistry and Molecular Biology, 1997Co-Authors: Francesco Pennacchio, Patrizia Falabella, Rocco Sordetti, Bradleigh S VinsonAbstract:Abstract The present study describes both the ultrastructural and the biochemical alterations induced by the parasitoid Cardiochiles Nigriceps Viereck on the prothoracic glands of the last (fifth) instar larvae of its host Heliothis virescens (F.). Gross morphological degeneration or signs of necrosis of the prothoracic gland cells were not observed. A rough endoplasmic reticulum more developed and electrondense than in non-parasitized controls was the only ultra-structural change occurring in the prothoracic glands of parasitized host last instar larvae. However, although structurally undegraded, 5 days after parasitoid oviposition into newly moulted host last (fifth) instar larvae, the prothoracic gland cells appeared to be functionally dead and became permeable to trypan blue. In contrast, cells of the prothoracic glands explanted on day 4 of the fifth instar, 3 days after parasitism, performed as above, were still alive and did not absorb the trypan blue, as observed in synchronous non-parasitized controls. These glands were used to study in vitro the major biochemical alterations at cellular level induced by the parasitoid. Ecdysteroid production, RNA synthesis, protein synthesis and phosphorylation were studied in response to crude prothoracicotropic hormone (PTTH) extract, forskolin and dibutyryl cAMP (dbcAMP), these latter compounds mimicking the PTTH action at different levels of the signal transduction pathway. The prothoracic glands of H. virescens last instar larvae, parasitized by C. Nigriceps , showed a severely depressed biosynthesis of ecdysteroids, which was not resumed with any of the stimulating treatments performed. This severely depressed biosynthetic activity was associated with a remarkable reduction in the rate of total protein synthesis and to a marked underphosphorylation of those proteins normally phosphorylated when healthy glands are challenged with dbcAMP. However, the early steps of the PTTH signal transduction pathway in the host prothoracic glands are apparently not altered by C. Nigriceps parasitism. This is supported by the identical effect of PTTH and dbcAMP treatments on RNA synthesis in the prothoracic glands of both parasitized and non-parasitized larvae. In fact, in both cases a reduced level of [ 3 H]uridine incorporation into the total RNA synthesized in vitro was observed in stimulated glands. These experimental data would suggest the occurrence of a parasitoid-induced block of protein synthesis translation in host prothoracic glands, partly determined by the marked underphosphorylation of key target regulatory proteins.
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morphology and ultrastructure of the serosal cells teratocytes in Cardiochiles Nigriceps viereck hymenoptera braconidae embryos
International Journal of Insect Morphology & Embryology, 1994Co-Authors: Francesco Pennacchio, S B Vinson, E. TremblayAbstract:Abstract The morphogenetic changes of the serosal membrane during embryonic development of Cardiochiles Nigriceps Viereck (Hymenoptera : Braconidae) were investigated. Eggs observed soon after oviposition into the natural host Heliothis virescens (F.) (Lepidoptera, Noctuidae) showed a transparent chorion and a uniform texture. After 5 hr, the embryo exhibited a distinct granular appearance and by 12 hr attained the germ band stage. A serosal membrane originated from the anterior pole of the embryo between 14 and 15 hr after the egg was laid, eventually forming with the cells both in the anterior and posterior pole a continuous envelope around the developing embryo. Ultrastructural observations revealed that the serosal cells in contact with the abdominal region of the embryo, beginning 24–25 hr after oviposition, formed a syncytium. However, the syncytial tissue did not extend to the cells around the head and thorax. The serosal cells at both embryo poles increased in size without losing their structural organization, and developed into teratocytes when the larva hatched. In contrast, the serosal cells surrounding the body of the embryo persisted longer on the head and thorax region of the newly hatched larva, while the syncytial tissue degraded more rapidly after hatching. In vitro rearing experiments showed that C. Nigriceps embryos removed from parasitized host larvae just before and just after serosa formation, hatched only when the medium used was formulated with the addition of fetal bovine serum. Embryos did not develop or hatch when placed in a serum-free medium. Once the syncytium deriving from the serosal membrane became evident, embryos readily developed and hatched in serumfree media. The results of this study seem to suggest that the serosal embryonic membrane could have a nutritional role for the developing parasitoid embryo.
Bradleigh S Vinson - One of the best experts on this subject based on the ideXlab platform.
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prothoracic gland inactivation in heliothis virescens f lepidoptera noctuidae larvae parasitized by Cardiochiles Nigriceps viereck hymenoptera braconidae
Journal of Insect Physiology, 1998Co-Authors: Francesco Pennacchio, P Varricchio, Patrizia Falabella, Rocco Sordetti, C Malva, Bradleigh S VinsonAbstract:Abstract Heliothis virescens (F.) last instar larvae parasitized by the endophagous braconid Cardiochiles Nigriceps Viereck fail to attain the pupal stage, due to a parasitoid-induced alteration of ecdysteroid biosynthesis and metabolism. Currently available information on host prothoracic gland inactivation in this host-parasitoid system is reported here. Prothoracic glands of H. virescens mature larvae show a depressed biosynthetic activity, without undergoing gross morphological disruption. The ultrastructure of gland cells is characterized by minor parasitoid-induced changes, with the rough endoplasmic reticulum appearing more developed and electrondense than in nonparasitized controls. Eventually, the cells of prothoracic glands of parasitized host last instar larvae die but maintain their structural integrity. The inactivation of pupally committed host prothoracic glands is achieved through the disruption of the PTTH signal transduction pathway. The second messenger cAMP appears to be normally produced in response to PTTH stimulation of glands explanted from parasitized host larvae, however the downstream activation of the cAMP-dependent protein kinase does not appear to occur. In fact, a marked underphosphorylation of regulatory target proteins is observed. This underphosphorylation is associated with a significant reduction in general protein synthesis, which appears to be blocked at the translational level, to a redirection of specific protein synthesis and to a drastic suppression of ecdysteroidogenesis. These parameters appeared to be correlated in a kinetic time-course study, confirming their functional link. C. Nigriceps polydnavirus (CnPDV) plays a major role in the inactivation of pupally committed host prothoracic glands, while putative factors occurring in the host haemolymph do not seem to be of particular importance at that developmental stage. Southern blot hybridization indicates the occurrence of PKI(protein kinase inhibitor)-like genes in the C. Nigriceps genome, which, in contrast, are undetectable in H. virescens.
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regulation of heliothis virescens prothoracic glands by Cardiochiles Nigriceps polydnavirus
Archives of Insect Biochemistry and Physiology, 1998Co-Authors: Francesco Pennacchio, Patrizia Falabella, Bradleigh S VinsonAbstract:Heliothis virescens (F.) Larvae parasitized by the endophagous braconid Cardiochiles Nigriceps Viereck fail to attain the pupal stage. This developmental alteration is caused by both an inactivation of prothoracic glands of last-instar larvae and an altered ecdysone metabolism. Decrease in ecdysteroidogenesis in vitro was already evident in glands explanted from larvae that have attained the early cell formation stage (day 4 of fifth instar), 6 h after parasitoid oviposition. Ecdysteroidogenesis nearly ceased by 24 h after parasitoid oviposition. The degree of this biosynthetic depression increased as the time between parasitization and gland dissection increased. A time-course study allowed us to determine if both the degree of phosphorylation of regulatory target proteins, the rate of general protein synthesis and ecdysteroidogenesis decreased in concert over time. The results provide further evidence in support of the hypothesis that these cellular activities in prothoracic gland cells are functionally correlated in steroidogenic responses. Treatment with calyx fluid and venom of C. Nigriceps duplicates the parasitism-induced inactivation of host prothoracic glands. A 6-h conditioning in vitro of pupally committed host prothoracic glands with these parasitoid female reproductive secretions resulted in a significant depression of their ecdysteroid production. However, glands lost their sensitivity to calyx fluid and venom treatment when explanted from hosts that had already attained the cell formation stage. This was further supported by the fact that nearly all the host larvae parasitized on day 4 of fifth instar (cell formation stage) pupated, while parasitization on day 3 resulted in only 11% pupation. The coupled trioxsalen/UV irradiation treatment of C. Nigriceps calyx fluid and venom eliminated their negative effect on biosynthetic activity in vitro by host prothoracic glands. This result indirectly demonstrates that C. Nigriceps polydnavirus is the major regulating factor involved in the host prothoracic gland inactivation. Arch. Insect Biochem. Physiol. 38:1–10, 1998. © 1998 Wiley-Liss, Inc.
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biochemical and ultrastructural alterations in prothoracic glands of heliothis virescens f lepidoptera noctuidae last instar larvae parasitized by Cardiochiles Nigriceps viereck hymenoptera braconidae
Insect Biochemistry and Molecular Biology, 1997Co-Authors: Francesco Pennacchio, Patrizia Falabella, Rocco Sordetti, Bradleigh S VinsonAbstract:Abstract The present study describes both the ultrastructural and the biochemical alterations induced by the parasitoid Cardiochiles Nigriceps Viereck on the prothoracic glands of the last (fifth) instar larvae of its host Heliothis virescens (F.). Gross morphological degeneration or signs of necrosis of the prothoracic gland cells were not observed. A rough endoplasmic reticulum more developed and electrondense than in non-parasitized controls was the only ultra-structural change occurring in the prothoracic glands of parasitized host last instar larvae. However, although structurally undegraded, 5 days after parasitoid oviposition into newly moulted host last (fifth) instar larvae, the prothoracic gland cells appeared to be functionally dead and became permeable to trypan blue. In contrast, cells of the prothoracic glands explanted on day 4 of the fifth instar, 3 days after parasitism, performed as above, were still alive and did not absorb the trypan blue, as observed in synchronous non-parasitized controls. These glands were used to study in vitro the major biochemical alterations at cellular level induced by the parasitoid. Ecdysteroid production, RNA synthesis, protein synthesis and phosphorylation were studied in response to crude prothoracicotropic hormone (PTTH) extract, forskolin and dibutyryl cAMP (dbcAMP), these latter compounds mimicking the PTTH action at different levels of the signal transduction pathway. The prothoracic glands of H. virescens last instar larvae, parasitized by C. Nigriceps , showed a severely depressed biosynthesis of ecdysteroids, which was not resumed with any of the stimulating treatments performed. This severely depressed biosynthetic activity was associated with a remarkable reduction in the rate of total protein synthesis and to a marked underphosphorylation of those proteins normally phosphorylated when healthy glands are challenged with dbcAMP. However, the early steps of the PTTH signal transduction pathway in the host prothoracic glands are apparently not altered by C. Nigriceps parasitism. This is supported by the identical effect of PTTH and dbcAMP treatments on RNA synthesis in the prothoracic glands of both parasitized and non-parasitized larvae. In fact, in both cases a reduced level of [ 3 H]uridine incorporation into the total RNA synthesized in vitro was observed in stimulated glands. These experimental data would suggest the occurrence of a parasitoid-induced block of protein synthesis translation in host prothoracic glands, partly determined by the marked underphosphorylation of key target regulatory proteins.
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alteration of ecdysone metabolism in heliothis virescens f lepidoptera noctuidae larvae induced by Cardiochiles Nigriceps viereck hymenoptera braconidae teratocytes
Insect Biochemistry and Molecular Biology, 1994Co-Authors: Francesco Pennacchio, Bradleigh S Vinson, E. Tremblay, Angela OstuniAbstract:Abstract The haemolymph titres of ecdysteroids in last instar Heliothis virescens (F.) larvae parasitized by Cardiochiles Nigriceps Viereck, or in larvae injected with teratocytes obtained from this parasitoid, were determined and compared to those of nonparasitized larvae. Ecdysteroids were extracted, purified by high performance liquid chromatography (HPLC), and the titres assessed by radioimmunoassay. The total ecdysteroid titre of parasitized day 5 of 5th instar larvae was consistently lower than in nonparasitized larvae of the same age. The main ecdysteroid inactivation products were both polar and C-26 hydroxylated compounds, which were detected as soon as the 20-hydroxyecdysone appeared to be produced. The subsequent increase of the total ecdysteroid titre registered on day 7 of 5th instar parasitized larvae was almost completely due to polar compounds, whereas 20-hydroxyecdysone remained at low levels. In contrast, the total ecdysteroid titre of nonparasitized H. virescens larvae was higher and inactive metabolites increased in concentration only the day before pupation, after the typical 20-hydroxyecdysone burst. When C. Nigriceps teratocytes, obtained from embryos hatched in vitro on a semi-defined medium, were injected into day 1 of nonparasitized host 5th instar larvae (new-slender stage), the total ecdysteroid titres on day 5 and 6 of these larvae were much higher than in nonparasitized controls. However, this high ecdysteroid titre was associated with low levels of 20-hydroxyecdysone and with a high titre of ecdysone and other unidentified ecdysteroids. The 20-hydroxyecdysone was the most abundant ecdysteroid released after enzymatic digestion of the polar ecdysteroids isolated from the haemolymph of parasitized day 7 of 5th instar larvae. In vitro incubation of teratocytes with radiolabelled 20-hydroxyecdysone resulted in the recovery of 34% of the total radioactivity in the HPLC fractions corresponding to polar metabolites. In contrast, only 4.5% of the total radioactivity was recovered in the same polar fractions when ecdysone was co-incubated with teratocytes. These results, along with those presented above, suggest that the inhibition of host pupation observed in parasitized H. virescens larvae is due in part to 20-hydroxyecdysone inactivation through the formation of polar metabolites and C. Nigriceps teratocytes seem to play an important role in this biochemical transformation.
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biochemical and developmental alterations of heliothis virescens f lepidoptera noctuidae larvae induced by the endophagous parasitoid Cardiochiles Nigriceps viereck hymenoptera braconidae
Archives of Insect Biochemistry and Physiology, 1994Co-Authors: Francesco Pennacchio, Bradleigh S Vinson, E. Tremblay, Toshiharu TanakaAbstract:All larval stages of Heliothis virescens (F.) parasitized by the endophagous larval parasitoid Cardiochiles Nigriceps Viereck, a braconid species belonging to the subfamily Microgasterinae, exhibit developmental arrest at last instar and fail to pupate. The major part of larval development of the parasitoid is synchronized with the arrested host last larval instar and the parasitoid first molt is never observed before the host attains the late digging stage. At this time, the total ecdysteroid titer of the hemolymph of parasitized hosts is very low and subsequently shows a slow and gradual increase, characterized by a low titer of 20-hydroxyecdysone (20-HE) associated with consistent amounts of inactive ecdysteroid polar metabolites. Juvenile hormone esterase (JHE) activity is high in both control and parasitized host larvae at the early digging stage of development, and juvenile hormone analogs (JHA) applied to parasitized host last instar larvae appear to suppress the parasitoid molt. Concurrent with these changes was an increase in the hemolymph titer of proteins which was maintained at a high level in parasitized larvae in contrast to the observed decrease in control larvae at the cell formation stage of development. Neck-ligation of newly molted host 5th instar parasitized larvae, prior to both JHE release and the increase in protein titers, inhibited growth and molting of the parasitoid. In contrast, ligation after JHE release and with high hemolymph protein titers resulted in parasitoid molting and growth. These data suggest that the host ecdysteroid hormones are not directly involved in the regulation of the parasitoid molt, although high juvenile hormone (JH) levels probably prevent it. More likely, molting is triggered by other biochemical changes, such as proteins or other factors occurring in the hemolymph. Molting of C. Nigriceps larvae in vitro into an ecdysone-free semidefined medium further supported the view that host ecdysone is not necessary for the molt. Teratocytes of C. Nigriceps seem to play an important role in the inactivation of 20-HE through its conversion to inactive polar metabolites, and along with female calyx fluid and venom which depress the secretory activity of the host prothoracic glands, they are the most important sources of host regulatory factors. © 1994 Wiley-Liss, Inc.
E. Tremblay - One of the best experts on this subject based on the ideXlab platform.
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alteration of ecdysone metabolism in heliothis virescens f lepidoptera noctuidae larvae induced by Cardiochiles Nigriceps viereck hymenoptera braconidae teratocytes
Insect Biochemistry and Molecular Biology, 1994Co-Authors: Francesco Pennacchio, Bradleigh S Vinson, E. Tremblay, Angela OstuniAbstract:Abstract The haemolymph titres of ecdysteroids in last instar Heliothis virescens (F.) larvae parasitized by Cardiochiles Nigriceps Viereck, or in larvae injected with teratocytes obtained from this parasitoid, were determined and compared to those of nonparasitized larvae. Ecdysteroids were extracted, purified by high performance liquid chromatography (HPLC), and the titres assessed by radioimmunoassay. The total ecdysteroid titre of parasitized day 5 of 5th instar larvae was consistently lower than in nonparasitized larvae of the same age. The main ecdysteroid inactivation products were both polar and C-26 hydroxylated compounds, which were detected as soon as the 20-hydroxyecdysone appeared to be produced. The subsequent increase of the total ecdysteroid titre registered on day 7 of 5th instar parasitized larvae was almost completely due to polar compounds, whereas 20-hydroxyecdysone remained at low levels. In contrast, the total ecdysteroid titre of nonparasitized H. virescens larvae was higher and inactive metabolites increased in concentration only the day before pupation, after the typical 20-hydroxyecdysone burst. When C. Nigriceps teratocytes, obtained from embryos hatched in vitro on a semi-defined medium, were injected into day 1 of nonparasitized host 5th instar larvae (new-slender stage), the total ecdysteroid titres on day 5 and 6 of these larvae were much higher than in nonparasitized controls. However, this high ecdysteroid titre was associated with low levels of 20-hydroxyecdysone and with a high titre of ecdysone and other unidentified ecdysteroids. The 20-hydroxyecdysone was the most abundant ecdysteroid released after enzymatic digestion of the polar ecdysteroids isolated from the haemolymph of parasitized day 7 of 5th instar larvae. In vitro incubation of teratocytes with radiolabelled 20-hydroxyecdysone resulted in the recovery of 34% of the total radioactivity in the HPLC fractions corresponding to polar metabolites. In contrast, only 4.5% of the total radioactivity was recovered in the same polar fractions when ecdysone was co-incubated with teratocytes. These results, along with those presented above, suggest that the inhibition of host pupation observed in parasitized H. virescens larvae is due in part to 20-hydroxyecdysone inactivation through the formation of polar metabolites and C. Nigriceps teratocytes seem to play an important role in this biochemical transformation.
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morphology and ultrastructure of the serosal cells teratocytes in Cardiochiles Nigriceps viereck hymenoptera braconidae embryos
International Journal of Insect Morphology & Embryology, 1994Co-Authors: Francesco Pennacchio, S B Vinson, E. TremblayAbstract:Abstract The morphogenetic changes of the serosal membrane during embryonic development of Cardiochiles Nigriceps Viereck (Hymenoptera : Braconidae) were investigated. Eggs observed soon after oviposition into the natural host Heliothis virescens (F.) (Lepidoptera, Noctuidae) showed a transparent chorion and a uniform texture. After 5 hr, the embryo exhibited a distinct granular appearance and by 12 hr attained the germ band stage. A serosal membrane originated from the anterior pole of the embryo between 14 and 15 hr after the egg was laid, eventually forming with the cells both in the anterior and posterior pole a continuous envelope around the developing embryo. Ultrastructural observations revealed that the serosal cells in contact with the abdominal region of the embryo, beginning 24–25 hr after oviposition, formed a syncytium. However, the syncytial tissue did not extend to the cells around the head and thorax. The serosal cells at both embryo poles increased in size without losing their structural organization, and developed into teratocytes when the larva hatched. In contrast, the serosal cells surrounding the body of the embryo persisted longer on the head and thorax region of the newly hatched larva, while the syncytial tissue degraded more rapidly after hatching. In vitro rearing experiments showed that C. Nigriceps embryos removed from parasitized host larvae just before and just after serosa formation, hatched only when the medium used was formulated with the addition of fetal bovine serum. Embryos did not develop or hatch when placed in a serum-free medium. Once the syncytium deriving from the serosal membrane became evident, embryos readily developed and hatched in serumfree media. The results of this study seem to suggest that the serosal embryonic membrane could have a nutritional role for the developing parasitoid embryo.
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biochemical and developmental alterations of heliothis virescens f lepidoptera noctuidae larvae induced by the endophagous parasitoid Cardiochiles Nigriceps viereck hymenoptera braconidae
Archives of Insect Biochemistry and Physiology, 1994Co-Authors: Francesco Pennacchio, Bradleigh S Vinson, E. Tremblay, Toshiharu TanakaAbstract:All larval stages of Heliothis virescens (F.) parasitized by the endophagous larval parasitoid Cardiochiles Nigriceps Viereck, a braconid species belonging to the subfamily Microgasterinae, exhibit developmental arrest at last instar and fail to pupate. The major part of larval development of the parasitoid is synchronized with the arrested host last larval instar and the parasitoid first molt is never observed before the host attains the late digging stage. At this time, the total ecdysteroid titer of the hemolymph of parasitized hosts is very low and subsequently shows a slow and gradual increase, characterized by a low titer of 20-hydroxyecdysone (20-HE) associated with consistent amounts of inactive ecdysteroid polar metabolites. Juvenile hormone esterase (JHE) activity is high in both control and parasitized host larvae at the early digging stage of development, and juvenile hormone analogs (JHA) applied to parasitized host last instar larvae appear to suppress the parasitoid molt. Concurrent with these changes was an increase in the hemolymph titer of proteins which was maintained at a high level in parasitized larvae in contrast to the observed decrease in control larvae at the cell formation stage of development. Neck-ligation of newly molted host 5th instar parasitized larvae, prior to both JHE release and the increase in protein titers, inhibited growth and molting of the parasitoid. In contrast, ligation after JHE release and with high hemolymph protein titers resulted in parasitoid molting and growth. These data suggest that the host ecdysteroid hormones are not directly involved in the regulation of the parasitoid molt, although high juvenile hormone (JH) levels probably prevent it. More likely, molting is triggered by other biochemical changes, such as proteins or other factors occurring in the hemolymph. Molting of C. Nigriceps larvae in vitro into an ecdysone-free semidefined medium further supported the view that host ecdysone is not necessary for the molt. Teratocytes of C. Nigriceps seem to play an important role in the inactivation of 20-HE through its conversion to inactive polar metabolites, and along with female calyx fluid and venom which depress the secretory activity of the host prothoracic glands, they are the most important sources of host regulatory factors. © 1994 Wiley-Liss, Inc.
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growth and development of Cardiochiles Nigriceps viereck hymenoptera braconidae larvae and their synchronization with some changes of the hemolymph composition of their host heliothis virescens f lepidoptera noctuidae
Archives of Insect Biochemistry and Physiology, 1993Co-Authors: Francesco Pennacchio, Bradleigh S Vinson, E. TremblayAbstract:Larval development of the parasitoid Cardiochiles Nigriceps Viereck occurs in the last instar larva of its host, Heliothis virescens (F.). This allows the parasitoid to exploit the nutritional increase in the biosynthetic activity occurring in the host in preparation for metamorphosis. To understand the biochemical basis of this host parasitoid developmental synchrony, we undertook host ligation studies and analyzed host hemolymph for proteins and glycerol esters. Parasitization affected the biochemical profile of the host. The hemolymph protein concentration of parasitized last instar H. virescens larvae increased through time, whereas unparasitized (control) larvae were characterized by a decrease in the protein titer when they reached the prepupal stage. The effect of parasitism on glyceride titers of host hemolymph was not as pronounced as the effect on proteins. Ligation conducted on 5th instar hosts, which were parasitized as 4th instars, affected parasitoid development in a time-dependent way. The percentage of successfully developing C. Nigriceps larvae increased with the increase of the time interval between parasitization and ligation. Ligation performed before day 2 of the 5th larval instar of H. virescens completely inhibited parasitoid development. Ligations that disrupted parasitoid developmentwere associated with a low host hernolymph protein concentration. Parasitoid development was successful when hernolymph protein titer was high, as occurred when ligations were performed after day 3 of the 5th host instar in both control and parasitized larvae. Ligations in both situations resulted in a slight increase in glyceride titers. The results suggest that host proteins and/or some factor(s) associated with them may play a role in parasitoid growth and development. © 1993 Wiley-Liss, Inc.
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Preliminary results on in vitro rearing of the endoparasitoid Cardiochiles Nigriceps from egg to second instar
Entomologia Experimentalis et Applicata, 1992Co-Authors: Francesco Pennacchio, S. Bradleigh Vinson, E. TremblayAbstract:The composition of an artificial medium and technical procedures used for in vitro rearing of the endophagous larval parasitoid Cardiochiles Nigriceps Viereck (Hymenoptera, Braconidae), from post-germ band egg to the 2nd instar larva, are described. Amino acids, carbohydrates, salts, and vitamins were supplied in defined amounts as an aqueous solution which, when supplemented with 20 mg/ml of bovine albumin, 5 mg/ml of lactalbumin (enzymatic hydrolysate), 20% (v/v) fetal bovine serum, 20% (v/v) milk and 10% (v/v) chicken egg yolk, allowed for parasitoid growth and molting to the 2nd instar. Molting to the final instar was never observed.
Rocco Sordetti - One of the best experts on this subject based on the ideXlab platform.
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Cardiochiles Nigriceps polydnavirus molecular characterization and gene expression in parasitized heliothis virescens larvae
Insect Biochemistry and Molecular Biology, 1999Co-Authors: P Varricchio, Patrizia Falabella, Rocco Sordetti, Franco Graziani, C Malva, Francesco PennacchioAbstract:Abstract Cardiochiles Nigriceps Viereck is an endophagous parasitoid of larval stages of the tobacco budworm, Heliothis virescens (F.). This hymenopteran parasitoid, belonging to the family Braconidae, is associated with a polydnavirus (CnPDV), injected at oviposition along with the egg. The infection of various tissues by CnPDV determines the suppression of the host immune system and the developmental arrest of mature host larvae. In this study, CnPDV has been characterized at the structural and molecular level. The negatively stained nucleocapsids show evident ‘end structures’ and a tail-like appendage. The CnPDV genome is typically segmented, with circular dsDNA molecules, ranging in size from 2.5 kb to more than 23 kb. The early expression pattern of CnPDV in parasitized hosts has been analysed and viral clones, genomic and cDNAs, identifying genes expressed within 48 h after parasitization have been isolated. The molecular organization of one of these genes, named CnPDV1, and its putative protein product have been determined. Significant sequence homologies with other known proteins were not detected. In situ hybridization experiments indicated that this gene is expressed in the prothoracic glands of parasitized host mature larvae. A functional analysis of CnPDV1 gene product is required to assess its possible role in the regulation of parasitoid-induced alterations of host larvae.
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prothoracic gland inactivation in heliothis virescens f lepidoptera noctuidae larvae parasitized by Cardiochiles Nigriceps viereck hymenoptera braconidae
Journal of Insect Physiology, 1998Co-Authors: Francesco Pennacchio, P Varricchio, Patrizia Falabella, Rocco Sordetti, C Malva, Bradleigh S VinsonAbstract:Abstract Heliothis virescens (F.) last instar larvae parasitized by the endophagous braconid Cardiochiles Nigriceps Viereck fail to attain the pupal stage, due to a parasitoid-induced alteration of ecdysteroid biosynthesis and metabolism. Currently available information on host prothoracic gland inactivation in this host-parasitoid system is reported here. Prothoracic glands of H. virescens mature larvae show a depressed biosynthetic activity, without undergoing gross morphological disruption. The ultrastructure of gland cells is characterized by minor parasitoid-induced changes, with the rough endoplasmic reticulum appearing more developed and electrondense than in nonparasitized controls. Eventually, the cells of prothoracic glands of parasitized host last instar larvae die but maintain their structural integrity. The inactivation of pupally committed host prothoracic glands is achieved through the disruption of the PTTH signal transduction pathway. The second messenger cAMP appears to be normally produced in response to PTTH stimulation of glands explanted from parasitized host larvae, however the downstream activation of the cAMP-dependent protein kinase does not appear to occur. In fact, a marked underphosphorylation of regulatory target proteins is observed. This underphosphorylation is associated with a significant reduction in general protein synthesis, which appears to be blocked at the translational level, to a redirection of specific protein synthesis and to a drastic suppression of ecdysteroidogenesis. These parameters appeared to be correlated in a kinetic time-course study, confirming their functional link. C. Nigriceps polydnavirus (CnPDV) plays a major role in the inactivation of pupally committed host prothoracic glands, while putative factors occurring in the host haemolymph do not seem to be of particular importance at that developmental stage. Southern blot hybridization indicates the occurrence of PKI(protein kinase inhibitor)-like genes in the C. Nigriceps genome, which, in contrast, are undetectable in H. virescens.
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biochemical and ultrastructural alterations in prothoracic glands of heliothis virescens f lepidoptera noctuidae last instar larvae parasitized by Cardiochiles Nigriceps viereck hymenoptera braconidae
Insect Biochemistry and Molecular Biology, 1997Co-Authors: Francesco Pennacchio, Patrizia Falabella, Rocco Sordetti, Bradleigh S VinsonAbstract:Abstract The present study describes both the ultrastructural and the biochemical alterations induced by the parasitoid Cardiochiles Nigriceps Viereck on the prothoracic glands of the last (fifth) instar larvae of its host Heliothis virescens (F.). Gross morphological degeneration or signs of necrosis of the prothoracic gland cells were not observed. A rough endoplasmic reticulum more developed and electrondense than in non-parasitized controls was the only ultra-structural change occurring in the prothoracic glands of parasitized host last instar larvae. However, although structurally undegraded, 5 days after parasitoid oviposition into newly moulted host last (fifth) instar larvae, the prothoracic gland cells appeared to be functionally dead and became permeable to trypan blue. In contrast, cells of the prothoracic glands explanted on day 4 of the fifth instar, 3 days after parasitism, performed as above, were still alive and did not absorb the trypan blue, as observed in synchronous non-parasitized controls. These glands were used to study in vitro the major biochemical alterations at cellular level induced by the parasitoid. Ecdysteroid production, RNA synthesis, protein synthesis and phosphorylation were studied in response to crude prothoracicotropic hormone (PTTH) extract, forskolin and dibutyryl cAMP (dbcAMP), these latter compounds mimicking the PTTH action at different levels of the signal transduction pathway. The prothoracic glands of H. virescens last instar larvae, parasitized by C. Nigriceps , showed a severely depressed biosynthesis of ecdysteroids, which was not resumed with any of the stimulating treatments performed. This severely depressed biosynthetic activity was associated with a remarkable reduction in the rate of total protein synthesis and to a marked underphosphorylation of those proteins normally phosphorylated when healthy glands are challenged with dbcAMP. However, the early steps of the PTTH signal transduction pathway in the host prothoracic glands are apparently not altered by C. Nigriceps parasitism. This is supported by the identical effect of PTTH and dbcAMP treatments on RNA synthesis in the prothoracic glands of both parasitized and non-parasitized larvae. In fact, in both cases a reduced level of [ 3 H]uridine incorporation into the total RNA synthesized in vitro was observed in stimulated glands. These experimental data would suggest the occurrence of a parasitoid-induced block of protein synthesis translation in host prothoracic glands, partly determined by the marked underphosphorylation of key target regulatory proteins.
Patrizia Falabella - One of the best experts on this subject based on the ideXlab platform.
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Cardiochiles Nigriceps polydnavirus molecular characterization and gene expression in parasitized heliothis virescens larvae
Insect Biochemistry and Molecular Biology, 1999Co-Authors: P Varricchio, Patrizia Falabella, Rocco Sordetti, Franco Graziani, C Malva, Francesco PennacchioAbstract:Abstract Cardiochiles Nigriceps Viereck is an endophagous parasitoid of larval stages of the tobacco budworm, Heliothis virescens (F.). This hymenopteran parasitoid, belonging to the family Braconidae, is associated with a polydnavirus (CnPDV), injected at oviposition along with the egg. The infection of various tissues by CnPDV determines the suppression of the host immune system and the developmental arrest of mature host larvae. In this study, CnPDV has been characterized at the structural and molecular level. The negatively stained nucleocapsids show evident ‘end structures’ and a tail-like appendage. The CnPDV genome is typically segmented, with circular dsDNA molecules, ranging in size from 2.5 kb to more than 23 kb. The early expression pattern of CnPDV in parasitized hosts has been analysed and viral clones, genomic and cDNAs, identifying genes expressed within 48 h after parasitization have been isolated. The molecular organization of one of these genes, named CnPDV1, and its putative protein product have been determined. Significant sequence homologies with other known proteins were not detected. In situ hybridization experiments indicated that this gene is expressed in the prothoracic glands of parasitized host mature larvae. A functional analysis of CnPDV1 gene product is required to assess its possible role in the regulation of parasitoid-induced alterations of host larvae.
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prothoracic gland inactivation in heliothis virescens f lepidoptera noctuidae larvae parasitized by Cardiochiles Nigriceps viereck hymenoptera braconidae
Journal of Insect Physiology, 1998Co-Authors: Francesco Pennacchio, P Varricchio, Patrizia Falabella, Rocco Sordetti, C Malva, Bradleigh S VinsonAbstract:Abstract Heliothis virescens (F.) last instar larvae parasitized by the endophagous braconid Cardiochiles Nigriceps Viereck fail to attain the pupal stage, due to a parasitoid-induced alteration of ecdysteroid biosynthesis and metabolism. Currently available information on host prothoracic gland inactivation in this host-parasitoid system is reported here. Prothoracic glands of H. virescens mature larvae show a depressed biosynthetic activity, without undergoing gross morphological disruption. The ultrastructure of gland cells is characterized by minor parasitoid-induced changes, with the rough endoplasmic reticulum appearing more developed and electrondense than in nonparasitized controls. Eventually, the cells of prothoracic glands of parasitized host last instar larvae die but maintain their structural integrity. The inactivation of pupally committed host prothoracic glands is achieved through the disruption of the PTTH signal transduction pathway. The second messenger cAMP appears to be normally produced in response to PTTH stimulation of glands explanted from parasitized host larvae, however the downstream activation of the cAMP-dependent protein kinase does not appear to occur. In fact, a marked underphosphorylation of regulatory target proteins is observed. This underphosphorylation is associated with a significant reduction in general protein synthesis, which appears to be blocked at the translational level, to a redirection of specific protein synthesis and to a drastic suppression of ecdysteroidogenesis. These parameters appeared to be correlated in a kinetic time-course study, confirming their functional link. C. Nigriceps polydnavirus (CnPDV) plays a major role in the inactivation of pupally committed host prothoracic glands, while putative factors occurring in the host haemolymph do not seem to be of particular importance at that developmental stage. Southern blot hybridization indicates the occurrence of PKI(protein kinase inhibitor)-like genes in the C. Nigriceps genome, which, in contrast, are undetectable in H. virescens.
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regulation of heliothis virescens prothoracic glands by Cardiochiles Nigriceps polydnavirus
Archives of Insect Biochemistry and Physiology, 1998Co-Authors: Francesco Pennacchio, Patrizia Falabella, Bradleigh S VinsonAbstract:Heliothis virescens (F.) Larvae parasitized by the endophagous braconid Cardiochiles Nigriceps Viereck fail to attain the pupal stage. This developmental alteration is caused by both an inactivation of prothoracic glands of last-instar larvae and an altered ecdysone metabolism. Decrease in ecdysteroidogenesis in vitro was already evident in glands explanted from larvae that have attained the early cell formation stage (day 4 of fifth instar), 6 h after parasitoid oviposition. Ecdysteroidogenesis nearly ceased by 24 h after parasitoid oviposition. The degree of this biosynthetic depression increased as the time between parasitization and gland dissection increased. A time-course study allowed us to determine if both the degree of phosphorylation of regulatory target proteins, the rate of general protein synthesis and ecdysteroidogenesis decreased in concert over time. The results provide further evidence in support of the hypothesis that these cellular activities in prothoracic gland cells are functionally correlated in steroidogenic responses. Treatment with calyx fluid and venom of C. Nigriceps duplicates the parasitism-induced inactivation of host prothoracic glands. A 6-h conditioning in vitro of pupally committed host prothoracic glands with these parasitoid female reproductive secretions resulted in a significant depression of their ecdysteroid production. However, glands lost their sensitivity to calyx fluid and venom treatment when explanted from hosts that had already attained the cell formation stage. This was further supported by the fact that nearly all the host larvae parasitized on day 4 of fifth instar (cell formation stage) pupated, while parasitization on day 3 resulted in only 11% pupation. The coupled trioxsalen/UV irradiation treatment of C. Nigriceps calyx fluid and venom eliminated their negative effect on biosynthetic activity in vitro by host prothoracic glands. This result indirectly demonstrates that C. Nigriceps polydnavirus is the major regulating factor involved in the host prothoracic gland inactivation. Arch. Insect Biochem. Physiol. 38:1–10, 1998. © 1998 Wiley-Liss, Inc.
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biochemical and ultrastructural alterations in prothoracic glands of heliothis virescens f lepidoptera noctuidae last instar larvae parasitized by Cardiochiles Nigriceps viereck hymenoptera braconidae
Insect Biochemistry and Molecular Biology, 1997Co-Authors: Francesco Pennacchio, Patrizia Falabella, Rocco Sordetti, Bradleigh S VinsonAbstract:Abstract The present study describes both the ultrastructural and the biochemical alterations induced by the parasitoid Cardiochiles Nigriceps Viereck on the prothoracic glands of the last (fifth) instar larvae of its host Heliothis virescens (F.). Gross morphological degeneration or signs of necrosis of the prothoracic gland cells were not observed. A rough endoplasmic reticulum more developed and electrondense than in non-parasitized controls was the only ultra-structural change occurring in the prothoracic glands of parasitized host last instar larvae. However, although structurally undegraded, 5 days after parasitoid oviposition into newly moulted host last (fifth) instar larvae, the prothoracic gland cells appeared to be functionally dead and became permeable to trypan blue. In contrast, cells of the prothoracic glands explanted on day 4 of the fifth instar, 3 days after parasitism, performed as above, were still alive and did not absorb the trypan blue, as observed in synchronous non-parasitized controls. These glands were used to study in vitro the major biochemical alterations at cellular level induced by the parasitoid. Ecdysteroid production, RNA synthesis, protein synthesis and phosphorylation were studied in response to crude prothoracicotropic hormone (PTTH) extract, forskolin and dibutyryl cAMP (dbcAMP), these latter compounds mimicking the PTTH action at different levels of the signal transduction pathway. The prothoracic glands of H. virescens last instar larvae, parasitized by C. Nigriceps , showed a severely depressed biosynthesis of ecdysteroids, which was not resumed with any of the stimulating treatments performed. This severely depressed biosynthetic activity was associated with a remarkable reduction in the rate of total protein synthesis and to a marked underphosphorylation of those proteins normally phosphorylated when healthy glands are challenged with dbcAMP. However, the early steps of the PTTH signal transduction pathway in the host prothoracic glands are apparently not altered by C. Nigriceps parasitism. This is supported by the identical effect of PTTH and dbcAMP treatments on RNA synthesis in the prothoracic glands of both parasitized and non-parasitized larvae. In fact, in both cases a reduced level of [ 3 H]uridine incorporation into the total RNA synthesized in vitro was observed in stimulated glands. These experimental data would suggest the occurrence of a parasitoid-induced block of protein synthesis translation in host prothoracic glands, partly determined by the marked underphosphorylation of key target regulatory proteins.