Progress 10/01/05 to 09/30/09
Outputs
OUTPUTS: This Hatch grant has supported three graduate students, two of which graduated with an M.S. and have taken positions in biotechnology firms (Monsanto and Invitrogen). Their training in this lab was instrumental in obtaining their positions. PARTICIPANTS: Seth Tauchman received his MS while working on this project and has joined the biotech Ag industry. This project provided him with a background and training to be hired. Jun Wang received her MS while working on this project and has joined the biotech industry. This project provided her with a background and training to be hired. Joliene Lindholm is the current student working on her Ph.D. TARGET AUDIENCES: Agricultural Biotechnology Industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The Hatch grant A Molecular Analysis of Juvenile Hormone Action had three scientific goals: (1) Determine how JH regulates hJHBP (a hemolymph JH transport protein) mRNA levels. (2) Determine putative response elements using functional or in vitro transcription assays. (3) Identify and sequence the response elements using mobility shift DNA binding assays and DNase footprinting analysis. We originally proposed to conduct our study on a hormone transport gene but ran into a number of technical difficulties. To find a more tractable gene, we turned to a microarray analysis which immediately led to several genes that proved interesting. We focused on one gene, Exchange Protein Activated by Cyclic AMP or Epac. When challenged with JH, expression of this gene rose nearly 4-fold over a 4 h period. Epac activates Rap1, a small GTPase, that is involved in altering cell shape and movement during development. It is important to note that the functions of Rap1 are in keeping with the morphostatic actions of JH on embryonic and larval cells. This is the first evidence of JH acting on a molecular pathway consistent with its developmental role. JH-regulated Epac transcriptional activity was also verified by qRT-PCR using an intron-specific probe. These data, in conjunction with exon-specific probes, strongly support the idea that JH does indeed up-regulate the expression of specific genes (goal 1). JH up-regulation was confirmed in vivo. Since this work was carried out in Drosophila, databases could be queried for potential response elements (goal 2). We located an element common between JH up-regulated genes in Drosophila and honey bees (18). This sequence was repeated 3 times in the upstream region within 2.5 kb of the start site. Most gratifying is the finding that the JH agonist methoprene had no effect on human Epac expression.
Publications
- Goodman, W.G. and Granger, N.A. 2005. The juvenile hormones. In Comprehensive Molecular Insect Science (ed. by L.I. Gilbert, K. Iatrou, S. Gill). Vol 3 pp. 319-408.
- Elsevier, Amsterdam Tauchman, S.J., Lorch, J.M., Orth, A.P., Goodman, W.G. 2007. Effects of stress on the hemolymph juvenile hormone binding protein titers of Manduca sexta. Insect Biochemistry and Molecular Biology. 37:847-854.
- Willis DK, Wang J, Lindholm JR, Orth AP, Goodman WG, 2008. Microarray analysis of juvenile hormone response in Drosophila melanogaster S2 cells. Journal of Insect Science (in press).
- Wang J., Lindholm, J.R., Willis, D.K., Orth, A.P., Goodman, W.G. 2009. Juvenile hormone regulation of Drosophila Epac - A guanine nucleotide exchange factor for Rap1 small GTPase. Mol. Cell. Endocrinol.305:30-7.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: From a practical standpoint, the JH analogs are used in pest insect control programs where the more toxic pesticides cannot be employed. Understanding how they act at the molecular level may offer insight into the development of new, safer insect control strategies. Because they are used in households, urban areas, medically sensitive areas and dairy farms, it becomes imperative to understand how these hormones act on nontarget organisms, particularly humans. From the output standpoint, two graduate students were trained and graduated with graduate degrees. A third graduate student is now involved in the project. To date, the project has generated three publications. PARTICIPANTS: S.J. Tauchman, Department of Entomology UW Madison MS degree; J. Wang, Department of Entomology UW Madison MS degree; J.R. Stanford, Department of Entomology MS degree in progress; D.K. Willis, USDA/ARS Vegetable Crops Research Unit UW Madison, A.P. Orth Genomics Institute of the Novartis Research Foundation, San Diego TARGET AUDIENCES: Pesticide industry Pest insect control industry Academics PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The objectives of this lab are to elucidate how an insect developmental hormone regulates gene expression at the molecular level. To take advantage of the power of genetic tools, we turned to Drosophila. We were unable to find a JH response element in the regions examined. The second model system employs the Drosophila gene, Epac (exchange protein directly activated by cyclic AMP). Our recent microarray studies indicate that like the hJHBP gene, Epac is expressed very rapidly when JH titers are elevated. We have turned to a Drosophila cell line which facilitates a simpler system in which to study the process. We have made significant progress in studying this JH-regulated gene using quantitative RT-PCR. We have now demonstrated that Epac is regulated in a dose-dependent fashion with the lowest dose to stimulate a significant response to be 100 ng/ml JH III. The first significant rise above background occurs at 2 h after hormone treatment. We have tested the homologs JH I, Bisepoxy JH III, (10R) JH III and (10S) JH III and the commercially available growth regulator, methoprene. All compounds increase Epac mRNA at about the same level and in the same tempororal sequence. JH acid, a product of hormone metabolism has no affect on the gene. Our discovery that JH alone can up-regulate a specific gene is highly significant; all other studies indicate that ecdysone, the molting hormone is necessary for JH action. Another significant finding is that JH does not up-regulate Epac expression in a Human cell line. This finding supports the contention that JH is safe for human use. Epac is involved in the RAP1 signal pathway that modifies cytoskeletal structure. Our preliminary studies indicate that cell adhesion is affected by JH which is in line with the action of RAP1. A molecular analysis of JH action is important for both basic and applied research. The central role of JH in orchestrating the insect life cycle and reproductive events has not gone unnoticed among agroecologists, chemists, and entomologists who are concerned with developing better strategies for insect pest management. The short half-life of the JH agonists in the environment and the apparent low vertebrate toxicity make these insect growth regulators attractive for controlling insect pests. The JH agonists were first proven effective in disrupting insect development better than three decades ago and they continue to play a role in the control of certain pest species in integrated pest management programs. Unfortunately, the discovery of laboratory-induced resistance to some JH agonists underscores the need to continue the search for new growth regulators. Expanding our knowledge of how this hormone acts at the molecular level will aid in designing more effective JH agonists and antagonists. Elucidating the mode of action of a commercially used pesticide is critical to its continued use. While the juvenile hormones appear relatively safe to vertebrates, there may be reasons for not using the compound that we are unsure of. Thus, one of the goals of this research is to verify the mode of action of the hormone at the molecular level to make sure it is safe.
Publications
- Wang, J., Lindholm, J.R., Willis, D.K., Orth, A.P. and Goodman, W.G. 2009 Juvenile hormone regulation of Drosophila Epac - a guanine nucleotide exchange factor for Rap1 Small GTPase. Molecular and Cellular Endocrinology(accepted for publication).
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Juvenile hormones (JH) are central to insect growth, development, and reproduction. Their phylogenetic restriction to arthropods has made the synthetic JH agonists an important tool in pest management. Despite better than 2 decades of extensive field use, the mode of action of these hormones is still unclear. The rationale for elucidating the molecular mechanism(s) of JH is three-fold. First, JH agonists are used to control certain agriculturally important insect pests and disease vectors. Elucidation of JH action at the molecular level is crucial to developing more effective agonists for pest suppression. Second, failure to understand how JH and its agonists exert their influence at the molecular level may have serious toxicological implications for humans and domestic livestock. A detailed molecular knowledge of how these molecules act is key to their continued use as "environmentally safe" insecticides. Third, the underlying molecular mechanism(s) are unknown.
Understanding how this hormone acts on transcription of a single gene will provide a paradigm by which other important JH-regulated genes may be studied. This proposal has three objectives. (1) Determine how JH regulates gene expression. (2) Determine putative response elements using functional or in vitro transcription assays. (3) Identify and sequence the response elements using mobility shift DNA binding assays and DNase footprinting analysis. The tangible outcomes of this study, such as pesticides or new methods of pest insect control, are derivative of this very basic study. Yet, basic studies at the molecular level are central to new and safer methods of insect pest control. Indeed, this work now supports the idea that at least one of the most potent JH agonists has no affect on Human cells at the molecular level. A molecular analysis of JH action is important for both basic and applied research. The central role of JH in orchestrating the insect life cycle and reproductive
events has not gone unnoticed among agroecologists, chemists, and entomologists who are concerned with developing better strategies for insect pest management. The short half-life of the JH agonists in the environment and the apparent low vertebrate toxicity make these insect growth regulators attractive for controlling insect pests. The JH agonists were first proven effective in disrupting insect development better than three decades ago and they continue to play a role in the control of certain pest species in integrated pest management programs. Unfortunately, the discovery of laboratory-induced resistance to some JH agonists underscores the need to continue the search for new growth regulators. Expanding our knowledge of how this hormone acts at the molecular level will aid in designing more effective JH agonists and antagonists. Elucidating the mode of action of a commercially used pesticide is critical to its continued use. While the juvenile hormones appear relatively safe to
vertebrates, there may be reasons for not using the compound that we are unsure of. Thus, one of the goals of this research is to verify the mode of action of the hormone at the molecular level to make sure it is safe.
PARTICIPANTS: D.K. Willis, USDA/ARS Vegetable Crops Research Unit University of Wisconsin J. Wang Department of Entomology UW Madison - Received MS degree June 2007 J.R. Stanford Department of Entomology UW Madison - Working on MS A.P. Orth Genomics Institute of the Novartis Research Foundation - San Diego
TARGET AUDIENCES: Target audience includes academics who are interested in insect endocrinology at the molecular level. Also interested is the pesticide industry who can gain insight into how to develop new insect growth regulators.
PROJECT MODIFICATIONS: The original project focused on Manduca sexta alone. Our inability to isolate JH response elements forced us to look at another JH up-regulated gene. This gene, Epac, is even more central to the mode of action of JH than the original work. The study indicates that JH may be signaling its molecular action via a second messenger.
Impacts
Two genes one from the lepidopteran, Manduca sexta and one from the dipteran, Drosophila melanogaster have been studied. Both insect orders contain species that are serious agricultural and medical pests. The target gene for Lepidoptera is the hemolymph juvenile hormone binding protein gene (hJHBP). The upstream region of the gene has been characterized and constructs of the region have been developed for a reporter assay to determine if JH acts directly on the gene. We were unable to find a JH response element in the regions examined. The second model system employs the Drosophila gene, Epac (exchange protein directly activated by cyclic AMP). Our recent microarray studies indicate that like the hJHBP gene, Epac is expressed very rapidly when JH titers are elevated. We have turned to a Drosophila cell line which facilitates a simpler system in which to study the process. We have made significant progress in studying this JH-regulated gene using quantitative RT-PCR. We
have now demonstrated that Epac is regulated in a dose-dependent fashion with the lowest dose to stimulate a significant response to be 100 ng/ml JH III. The first significant rise above background occurs at 2 h after hormone treatment. We have tested the homologs JH I, Bisepoxy JH III, (10R) JH III and (10S) JH III and the commercially available growth regulator, methoprene. All compounds increase Epac mRNA at about the same level and in the same tempororal sequence. JH acid, a product of hormone metabolism has no affect on the gene. Our discovery that JH alone can up-regulate a specific gene is highly significant; all other studies indicate that ecdysone, the molting hormone is necessary for JH action. Another significant finding is that JH does not up-regulate Epac expression in a Human cell line. This finding supports the contention that JH is safe for human use. Epac is involved in the RAP1 signal pathway that modifies cytoskeletal structure. Our preliminary studies indicate that
cell adhesion is affected by JH which is in line with the action of RAP1.
Publications
- Willis, D.K., Wang, J., Stanford, J.R., Orth, A.P. and Goodman, W.G. (2007) Microarray Analysis of Juvenile Hormone Response in Drosophila melanogaster S2 cells. Journal of Insect Science (submitted)
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Progress 01/01/06 to 12/31/06
Outputs
Juvenile hormones (JH) are central to insect growth, development, and reproduction. Their phylogenetic restriction to arthropods has made the synthetic JH agonists an important tool in pest management. Despite better than 2 decades of extensive field use, the mode of action of these hormones is still unclear. The rationale for elucidating the molecular mechanism(s) of JH is three-fold. First, JH agonists are used to control certain agriculturally important insect pests and disease vectors. Elucidation of JH action at the molecular level is crucial to developing more effective agonists for pest suppression. Second, failure to understand how JH and its agonists exert their influence at the molecular level may have serious toxicological implications for humans and domestic livestock. A detailed molecular knowledge of how these molecules act is key to their continued use as "environmentally safe" insecticides. Third, the underlying molecular mechanism(s) are unknown.
Understanding how this hormone acts on transcription of a single gene will provide a paradigm by which other important JH-regulated genes may be studied. This proposal has three objectives. (1) Determine how JH regulates gene expression. (2) Determine putative response elements using functional or in vitro transcription assays. (3) Identify and sequence the response elements using mobility shift DNA binding assays and DNase footprinting analysis. Two candidate genes will be studied, a gene from the lepidopteran, Manduca sexta and a gene from the dipteran, Drosophila melanogaster. Both orders contain insects species that are serious agricultural and medical pests. The target gene for lepidoptera is the hemolymph juvenile hormone binding protein gene. This gene has been well studied and characterized. The upstream region of the gene has been characterized and constructs of the region have been developed for a reporter assay to determine if JH acts directly on the gene. The second model
system employs the Drosophila gene, Epac. Our recent microarray studies indicate that like the hJHBP gene, this gene is expressed very rapidly when JH titers are elevated. We have turned to a Drosophila cell line which facilitates a simpler system in which to study the process. While these studies have just started, we have made significant progress in studying this JH-regulated gene using quantitative RT-PCR. We have now demonstrated that the gene is regulated in a dose-dependent fashion with the lowest dose to stimulate a significant response to be 100 ng/ml JH III. The first significant rise above background occurs at 2 h after hormone treatment. We have tested the homologs JH I, Bisepoxy JH III, (10R) JH III and (10S) JH III and the commercially available growth regulator, methoprene. All compounds increase Epac mRNA at about the same level and in the same tempororal sequence. Epac is involved in the RAP1 signal pathway that modifies cytoskeletal structure. Our preliminary studies
indicate that cell adhesion is affected by JH which is in line with the action of RAP1.
Impacts
Elucidating the mode of action of a commercially used pesticide is critical to its continued use. While the juvenile hormones appear relatively safe to vertebrates, there may be reasons for not using the compound that we are unsure of. Thus, one of the goals of this research is to verify the mode of action of the hormone at the molecular level to make sure it is safe.
Publications
- S.J. Tauchman, Lorch, J.M., Orth, A.P., W.G. Goodman. 2007. Effects of Stress on the Hemolymph Juvenile Hormone Binding Protein Titers of Manduca sexta. Insect Biochem Molec. Biol. (in press).
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Progress 01/01/05 to 12/31/05
Outputs
Juvenile hormones (JH) are central to insect growth, development, and reproduction. Their phylogenetic restriction to arthropods has made the synthetic JH agonists an important tool in pest management. Despite better than 2 decades of extensive field use, the mode of action of these hormones is still unclear. The rationale for elucidating the molecular mechanism(s) of JH is three-fold. First, JH agonists are used to control certain agriculturally important insect pests and disease vectors. Elucidation of JH action at the molecular level is crucial to developing more effective agonists for pest suppression. Second, failure to understand how JH and its agonists exert their influence at the molecular level may have serious toxicological implications for humans and domestic livestock. A detailed molecular knowledge of how these molecules act is key to their continued use as environmentally safe insecticides. Third, the underlying molecular mechanism(s) are unknown.
Understanding how this hormone acts on transcription of a single gene will provide a paradigm by which other important JH-regulated genes may be studied. This proposal has three objectives. (1) Determine how JH regulates hJHBP mRNA levels. (2) Determine putative response elements using functional or in vitro transcription assays. (3) Identify and sequence the response elements using mobility shift DNA binding assays and DNase footprinting analysis. Two candidate genes will be studied, a gene from the lepidopteran, Manduca sexta and a gene from the dipteran, Drosophila melanogaster. Both orders contain insects species that are serious agricultural and medical pests. The target gene for lepidoptera is the hemolymph juvenile hormone binding protein gene. This gene has been well studied and characterized. The upstream region of the gene has been characterized and constructs of the region have been developed for a reporter assay to determine if JH acts directly on the gene. The second
model system employs the Drosophila gene, Cap. Our recent microarray studies indicate that like the hJHBP gene, this gene is expressed very rapidly when JH titers are elevated. We have turned to a Drosophila cell line which facilitates a simpler system in which to study the process. These studies have just started.
Impacts
A molecular analysis of JH action is important for both basic and applied research. The central role of JH in orchestrating the insect life cycle and reproductive events has not gone unnoticed among agroecologists, chemists, and entomologists who are concerned with developing better strategies for insect pest management. The short half-life of the JH agonists in the environment and the apparent low vertebrate toxicity make these insect growth regulators attractive for controlling insect pests. The JH agonists were first proven effective in disrupting insect development better than three decades ago and they continue to play a role in the control of certain pest species in integrated pest management programs. The discovery of laboratory-induced resistance to some JH agonists underscores the need to continue the search for new growth regulators. Expanding our knowledge of how this hormone acts at the molecular level will aid in designing more effective JH agonists and
antagonists.
Publications
- No publications reported this period
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