Progress 01/01/05 to 12/31/05
Outputs
1. To monitor the susceptibility of the Southwestern corn borer (SWCB) to Bacillus thuringiensis (Bt) toxins in transgenic corn, eight field SWCB populations were collected from TX, KS, MO, KY and TN in 2005, and reared in the lab for feeding bioassay. Feeding bioassay was used to determine LC50, EC50 and diagnostic concentration of two Bt toxins (Cry1Ab and Cry1F)on 8 field populations and 1 lab colony of SWCB. Although significant variation was observed among geographically distinct populations of SWCB in their susceptibility to Bt toxins,all field-collected populations were more susceptible to Cry1Ab toxin than the MS-lab colony. This is the sixth year that the susceptibility of SWCB to BT toxins was bioassayed in this lab and the result was reported to EPA via Agricultural Biotechnology Stewardship Technical Committee (ABSTC). So far no sign of SWCB resistance to Bt toxins was detected from the field-collected populations. This project is supported by ABSTC,
including Monsanto, Dow AgroSciences, Pioneer Hibred-Dupont, Aventis and Syngenta. 2. We have shown both EcR and USP are phosphoproteins and their phosphorylation is regulated by protein kinase C (PKC). Inhibition of PKC by specific PKC inhibitors blocked EcR and USP phosphorylation, resulting in the selective inhibition of the expression of 20E-response gene and protein. Preliminary results indicate that PKC-mediated EcR and USP phosphorylation regulates subcellular localization of EcR and USP. 3. We have purified a 56 kDa neuropeptide (p56) from Drosophila and obtained the p56 sequence information based on MALDI-TOF and Q-TOF data. Developmental profiles of the p56 from larvae, pupae and adult of the fruit fly and house fly were obtained using Western blot and immunocytochemistry approaches. Functional chracterization of the p56 using RNAi and baculovirus expressed p56 is underway. A postdoctoral fellow and a graduate student were recruted for the project in 2005. This project is
funded by NSF (3/1/2005-2/28/2008).
Impacts
1. Resistance management strategies to delay the onset of resistance of SWCB to Bt in transgenic crop depend on the effectiveness of resistance monitoring program capable of early detection of resistance that allows selected management strategies to be implemented before control failure occurs. The data from this project will provide basic information for selection of the best management strategies for insect pest control in transgenic cornfield. 2. Transformation from larvae to adult is one of the miracles of insect biology. This transformation is regulated mainly by steroid molting hormone 20 hydroxyecdysone (20E) via ecdysone receptor (EcR)/ultraspiracle protein (USP) complex. Understanding of the mechanism regulating the EcR/USP complex may provide information for designing more efficient ecdysone agonists as pesticides. The EcR/USP complex also has a potential to be used as gene switch for expressing insecticidal proteins or pharmaceutical peptides in plants. 3.
Preliminary data from the housefly and the fruit fly suggested that the 56 kDa protein is a novel neuropeptide and its role is postulated to be associated with the event at or post eclosion. Identification of the role and signaling pathway of the p56 neuropeptide would add an important body of knowledge towards understanding the process of insect growth and development. The information generated from this project can be used for designing more efficient insect control agents to disrupt the physiological process regulated by the p56 neuropeptide.
Publications
- Jin, X., Sun, X. and Song, Q. 2005. Woc gene mutation causes 20E-dependent alpha-tubulin detyrosination in Drosophila melanogaster. Arch. Insect Biochem. Physiol. 60: 116-129.
- Song, Q. and Sun, X. 2005. Hormonal control of insect growth and development. In Entomological Research: Progress and Perspective. Tong-Xian Liu and Kang Le [Ed]. Science and Technology Press, pp 89-123.
- Sun, X. and Song, Q. 2006. PKC-mediated USP phosphorylation is required for 20E-induced gene expression in the salivary glands of Drosophila melanogaster. Arch. Insect Biochem. Physiol. (In press)
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Progress 01/01/04 to 12/31/04
Outputs
1. To monitor the susceptibility of the Southwestern corn borer (SWCB) to Bacillus thuringiensis (Bt) toxins in transgenic corn, seven field SWCB populations were collected from TX, KS, MO, KY and TN, and reared in the lab for feeding bioassay. Feeding bioassay was used to determine LC50, EC50 and diagnostic concentration of two Bt. toxins on 7 field populations and 1 lab colony of SWCB. So far no sign of SWCB resistance to Bt toxins was detected from the field populations.This project is supported by Agricultural Biotechnology Stewardship Technical Committee (ABSTC) including Monsanto, Dow AgroSciences, Pioneer Hibred-Dupont, Aventis and Syngenta. 2. We have shown both EcR and USP are phosphoproteins and their phosphorylation is regulated by protein kinase C (PKC). Inhibition of PKC by specific PKC inhibitors blocked EcR and USP phosphorylation, resulting in the selective inhibition of 20E-response gene expression. 3. We have purified a 56 kDa neuropeptide (p56) from
Drosophila and obtained the p56 sequence information based on MALDI-TOF and Q-TOF data. Functional identification of the p56 using RNAi and baculovirus expressed p56 is underway. This project is recently funded by NSF.
Impacts
1. Resistance management strategies to delay the onset of resistance of SWCB to Bt depend on the effectiveness of resistance monitoring program capable of early detection of resistance that allows selected management strategies to be implemented before control failure occurs. The data from this project will provide basic information for selection of the best management strategies for insect pest control in transgenic cornfield. 2. Transformation from larvae to adult is one of the miracles of insect biology. This transformation is regulated mainly by steroid molting hormone 20 hydroxyecdysone (20E) via ecdysone receptor (EcR)/ultraspiracle protein (USP) complex. Understanding of the mechanism regulating the EcR/USP complex may provide information for designing more efficient ecdysone agonists as pesticides. The EcR/USP complex also has a potential to be used as gene switch for expressing insecticidal proteins or pharmaceutical peptides in plants. 3. Preliminary data
from the housefly and the fruit fly suggested that the 56 kDa protein is a novel neuropeptide and its role is postulated to be associated with the event at or post eclosion. Identification of the role and signaling pathway of the p56 neuropeptide would add an important body of knowledge towards understanding the process of insect growth and development. The information generated from this project can be used for designing more efficient insect control agents to disrupt the physiological process.
Publications
- Sun X., Barrett B.A. and Song Q. (2004) Effects of age and length of exposure on the reproduction of adult Codling moth exposed to surfaces treated with ecdysone. J Entomol Sci 39, 417-425.
- Liu X., Tanaka Y., Song Q., Xu B., Hua Y. (2004) Bombyx mori prothoracicostatic peptide inhibits ecdysteroidogenesis in vivo. Arch Insect Biochem Physiol 56, 155-61.
- Jin X., Sun X. and Song Q. (2005) Woc gene mutation causes 20E-dependent a-tubulin detyrosination in Drosophila melanogaster. Arch Insect Biochem Physiol (accepted).
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Progress 01/01/03 to 12/31/03
Outputs
1. To monitor the susceptibility of the Southwestern corn borer (SWCB) to Bacillus thuringiensis (Bt) toxins in transgenic corn, six field SWCB populations were collected from TX, KS, MO, KY and TN, and reared in the lab for feeding bioassay. Feeding bioassay was used to determine LC50, EC50 and diagnostic concentration of three Bt. toxins on 6 field populations and 1 lab colony of SWCB. So far no sign of SWCB resistance to Bt toxins was detected from the field populations. 2. We have confirmed that Drosophila ultraspiracle protein (USP) is a phosphoprotein and its phosphorylation is regulated by protein kinase C (PKC). Inhibition of PKC by specific PKC inhibitors blocked USP phosphorylation, resulting in the inhibition of 20E-response gene expression. We generated phosphorylation site-specific usp mutants by site-directed mutagenesis in collaboration with Dr. Vince Henrich (UNC-Greensboro). These usp mutants have been inserted into transfer vector and are in the
process of expressing them for functional study in baculovirus expression system. 3. We have characterized a 56 kDa neuropeptide (p56) from Drosophila using a p56 specific monoclonal antibody and collected over 60 grams of Drosophila heads for purification of the p56 neuropeptide using combination of ammonium sulfate precipitation, ion exchange and size exclusive chromatography, and 2D gel electrophoresis. The purified p56 peptide will be subjected to partial amino acid sequencing.
Impacts
1. Resistance management strategies to delay the onset of resistance of SWCB to Bt depend on the effectiveness of resistance monitoring program capable of early detection of resistance that allows selected management strategies to be implemented before control failure occurs. The data from this project will provide basic information for selection of the best management strategies for insect pest control in transgenic cornfield. 2. Transformation from larvae to adult is one of the miracles of insect biology. This transformation is regulated mainly by steroid molting hormone 20 hydroxyecdysone (20E) via ecdysone receptor (EcR)/ultraspiracle protein (USP) complex. Understanding of the mechanism regulating the EcR/USP complex may provide information for designing more efficient ecdysone agonists as pesticides. The EcR/USP complex also has a potential to be used as gene switch for expressing insecticidal proteins or pharmaceutical peptides in plants. 3. Preliminary data
from the housefly and the fruit fly suggested that the 56 kDa protein is a novel neuropeptide and its role is postulated to be associated with the event at or post eclosion. Identification of the role and signaling pathway of the p56 neuropeptide would add an important body of knowledge towards understanding the process of insect growth and development. The information generated from this project can be used for designing more efficient insect control agents to disrupt the physiological process.
Publications
- Liu X., Chen X., Xu Q., Xu B., Yoshiaki T., Song Q. and Hua Y.(2004)Biological activities of analogs of Bombyx mori prothoracicostatic peptide and the relationship with their structures. Arch. Insect Biochem. Physiol (Accepted).
- Sun X., Barrett B.A. and Song Q. (2004) Effects of age and length of exposure on the reproduction of adult Codling moth exposed to surfaces treated with ecdysone. J. Entomol. Sci. (In press).
- Sun X., Song Q. and Barrett B.A. (2003) Effect of ecdysone agonists on the expression of EcR, USP and other specific proteins in the ovaries of Codling moth (Cydia pomonella). Insect Biochem. Molec. Biol. 33, 829-840.
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Progress 01/01/02 to 12/31/02
Outputs
1. We have confirmed Drosophila ultraspiracle protein (USP) as a phosphoprotein and generated phosphorylation site-specific usp mutants using site-directed mutagenesis in collaboration with Dr. Vince Henrich (UNC-Greensboro). The open reading frames of these phosphorylation site-specific usp mutants have been cloned into pBlueBac4.5 transfer vector and are in the process of expressing them in Sf9 cell line using baculovirus expression system for functional study. 2. Six field SWCB populations were collected from TX, KS, MO, KY and TN, and reared them in the lab for the assay. Feeding bioassay was used to determine LC50, EC50 and diagnostic concentration of 3 Bt toxins on 6 field populations and 1 lab colony of SWCB. The data indicated that no significant changes in the susceptibility of SWCB to Bt toxins have been found.
Impacts
Insect molting and metamorphosis are regulated by ecdysteroids via ecdysone receptor (EcR) and its heterodimer, ultraspiracle protein (USP). Both EcR and USP have been demonstrated to be phosphoproteins and phosphorylation has been implicated in regulating the ligand and perhaps DNA binding activities of the EcR/USP complex. Thus protein phosphorylation provides another layer of regulatory mechanism mediating the function of the EcR/USP complex. The goal of the proposed research is to identify the specific phosphorylation sites of USP protein and to investigate the role of USP phosphorylation in the function of the EcR/USP complex as a first step toward future studies of the mechanisms governing the structure and function of the EcR/USP complex. Understanding of the roles of protein phosphorylation in modulating the function of the EcR/USP complex, the signaling pathway(s) that regulating EcR and USP phosphorylation, and the transcriptional events regulated by the
EcR/USP via phosphorylation may provide information for designing more efficient control agents that disrupt normal molting process.
Publications
- Sun X., Song Q. and Barrett B. (2003) Effects of Ecdysone Agonists on the Expression of EcR, USP and Other Specific Proteins in the Ovaries of the Codling Moth (Cydia pomonella). Insect Biochem. Molec. Biol. (in press).
- Xiaoping Sun, Qisheng Song and Bruce Barrett (2003) Effect of Ecdysone Agonists on Vitellogenesis and the Expression of EcR and USP in Codling Moth (Cydia pomonella). Arch. Insect Biochem. Physiol. 52, 115-129.
- Song Q., Sun X. and Jin X. (2003) 20E-regulated USP phosphorylation in Drosophila melanogaster. Insect Biochem. Molec. Biol. (in press).
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Progress 01/01/01 to 12/31/01
Outputs
In 2001, We were trying to determine the impact of phosphorylation on EcR/USP complex function by making mutations on potential phosphorylation sites of USP using site-directed mutagenesis as approach. This is still in the progress. We also investigated the roles of ecdysone-suppressed proteins in the ecdysone-deficient mutant of Drosophilla melanogaster. We continued our effort in determining the susceptibility of the southwestern corn borer (SWCB) to Bacillus thuringienesis (Bt.) toxins. LC50 and EC50 values at 7 and 14 days after treatment (feeding bioassay) were determined for 6 field populations from the midwest region and 1 lab colony.
Impacts
EcR/USP complex mediates 20-hydroxyecdysone-regulated insect molting and metamorphosis. Understanding of mechanisms that govern the function of EcR/USP complex may lead to develop better insect control strategies by using insect growth regulators or their agonists or antagonists as control agents. Monitoring the susceptibility of SWCB to Bt. will provide information for better resistance management strategies to delay the onset of resistance to Bt.
Publications
- No publications reported this period
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Progress 01/02/00 to 12/31/00
Outputs
In 2000, the focus was on the characterization of ultraspiracle protein (USP), the heterodimer partner of ecdysone receptor(EcR). Three USP monoclonal antiboty-immnuoreactive protein bands with molecular size of 54, 52, and 48 kDa (refered as p54, p52, and p48) were identified in Drosophila, showing distinct tissue and temporal specific expression profiles. p54 has been demonstrated to be the phosphorylated form of p52. The identity of p48 remains unclear. The role of USP phosphorylation in regulating the function of EcR/USP complex is under investigation. The susceptibility of the southwestern corn borer (SWCB) to Bacillus thuringienesis (Bt.) toxins was examined. LC50 and EC50 values at 7 and 14 days after treatment (feeding bioassay) were determined for 7 field populations and 1 lab colony. The bioassay using growth inhibition is considered to be more sensitive in determining the susceptibility of the SWCB to Bt. toxins tested.
Impacts
EcR/USP complex mediates 20-hydroxyecdysone-regulated insect molting and metamorphosis. Understanding of mechanisms that govern the function of EcR/USP complex may lead to develop better insect control strategies by using insect growth regulators or their agonists or antagonists as control agents. Monitoring the susceptibility of SWCB to Bt. will provide information for better resistance management strategies to delay the onset of resistance to Bt.
Publications
- 1. Gilbert L.I., Rybczynski R., Song Q., Mizoguchi A., Morreale R., Smith W.A., Matubayashi H., Shionoya M., Nagata S. and Kataoka H. (2000) Dynamic regulation of prothoracic gland ecdysteroidogenesis: Manduca sexta recombinant prothoracicotropic hormone and brain extracts have identical effects. Insect Biochem. Molec. Biol. 30, 1079-1089.
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