Progress 10/01/03 to 09/30/04
Outputs
Specific aims: The flesh fly, Sarcophaga crassipalpis, overwinters in a facultative pupal diapause. We reported that the developmental stasis observed during flesh fly diapause may be linked to a G1 cell cycle arrest, that is characterized by a decrease in pcna expression. We're therefore examining the molecular regulation of this model in order to find potential targets of intervention to control the diapause program in insect pest species. With this in mind, our specific aims are to: 1. further define the role of PCNA in Sarcophaga diapause. 2. study the role of other G1/S phase genes in diapause. 3. study the cell cycle genes in an alternate insect model of diapause. 4. define the events necessary for diapause programming during the photosensitive stage. 5. elucidate the signal transduction events associated with diapause termination. Results: During year two of the granting period, we focused our efforts on aims 2 and 3. The following will be a brief description of
the progress made on each aim. Data acquired from these experiments resulted in two publications and two manuscripts in preparation. Aim 2. We have generated clones of several cell cycle regulatory genes from the flesh fly, however we focused our efforts on two genes, cyclin e and the transcription factor e2f1. RT-PCR has yielded data suggesting that both of these genes are down-regulated during flesh fly diapause, thus controlling the transcriptional repression of the pcna gene during diapause. cyclin e levels decrease by 2 days after diapause entrance, while pcna levels are not down-regulated until 14 days after the initiation of diapause. We have also constructed double-stranded RNAs against all three gene products to use in RNA intereference experiments to elucidate the mechanism by which the down-regulation of these gene products might control the developmental arrest associated with insect diapause. Aim 3. While cell cycle regulation may play a role in flesh fly diapause, we
have not been able to support this finding in our alternate diapause model, the tobacco hornworm. While it also diapauses during the pupal stage, no down-regulation of proliferation genes are obsereved during hornworm diapause. Therefore, an alternate approach to study diapause-regulation in this Lepidopteran species has been initiated. High-throughput 2D-PAGE has yielded several proteins that are differentially expressed during the diapause program in the hornworm. To date, we have isolated 7 proteins that are diapause-specific and 8 that are non-diapause-specific. MALDI-TOF will be used to identify these proteins. Plans for the Coming Year: In year four, we will focus primarily on the mechanism(s) of the gene products during the diapause program in flesh flies. Specifically, we will examine gene expression at the initiation and termination of diapause in the flesh fly and use RNAi or antisense oligonucleotides to control the production of specific diapause-associated protein
products. We will also use MALDI-TOF and/or LC/MS/MS to identify the proteins that are differentially produced in diapausing and non-diapausing tobacco hornworms.
Impacts
Specific information on the molecular regulation of insect dormancy may yield novel strategies for the control of insect pests. Our results suggest that the control of cell cycle genes might be specieis specific as it appears that the tobacco hornworm does not control their diapause state by inhibiting cell proliferation.
Publications
- Hayward, S.A., S. Pavlides, J. Reinhart, S.P. Tammariello, and D.L. Denlinger. 2005. Temporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through diapause quiescence. (Journal of Insect Physiology- in press)
- Pavlides, S, K. Weir and S.P. Tammariello. 2004. Live and Let Diapause: Cell Cycle Regulation During Insect Overwintering. In: Life in the Cold. Evolution, Mechanisms, Adaptation and Application. Institute of Arctic Biology. 293-298.
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Progress 10/01/02 to 09/30/03
Outputs
During year two of the granting period, we made significant strides to answer specific aims1, 2 and 4. Data acquired from these experiments will result in the submission of three peer-reviewed manuscripts in late 2003 or early 2004. Aim 1. We previously reported that this gene was differentially expressed throughout the diapause program in the flesh fly, Sarcophaga crassipalpis. Semi-quantitative RT-PCR and quantitative Real-Time PCR were performed to determine the expression pattern of this gene product during diapause initiation, maintainence and termination in the flesh fly. Results acquired from these experiments revealed that PCNA expression is basal during the entire period of pupal diapause, followed by a15-fold transcriptional up-regulation within 24 hours after the termination of diapause. In situ hybridization using laser scanning confocal microscopy verified these data and further defined the spatial distribution of this gene product in brains following
diapause termination in flesh flies. We also have examined the expression of the PCNA gene product during long-day (non-diapause) development. We are focusing our efforts on the transcriptional control of PCNA at the day two- and day three-post pupation boundary between long-day and short-day-reared flies. This is the period when the physiological differences, such as developmental stasis and decrease in metabolism, become most apparent in short day-reared flies. To further understand the mechanistic role that PCNA plays in development, we attempted RNA interference (RNAi) using small antisense oligonucleotides that are complementary to the termini of the PCNA mRNA sequence. Preliminary results suggest that normal development is inhibited following injection of antisense PCNA in the larvae and pupae. Specifically, we observe that the antisense-treated organisms fail to develop past the early pupal stage. These data suggest that the PCNA gene product is necessary for continuous
development in flesh flies. Aim 2. We have generated clones of several cell cycle regulatory genes from the tobacco hornworm, in order to study the regulation of diapause in an alternate, and more agriculturally relevant, insect species. Portions of the hornworm cyclin E, and dacapo genes have been isolated and sequenced in my lab. The transcriptional regulation of these genes should yield significant data to better understand the control of diapause in different insect orders. Aim 4. To address the components necessary for diapause programming in flesh flies, we cloned a portion of the blue light photoreceptor cryptochrome from S. crassipalpis. Diapause is programmed during an embryonic photosensitive stage in which the blue-light photoperiod is measured by brain cells of the embryos while still in the mother's uterus. The clone was generated using degenerative PCR oligonucleotide primers generated against Drosophila melanogaster cryptochrome. Subsequently, RNA interference (RNAi)
was attempted to abolish the production of this gene product, however we have not yet been able to define the strict protocol for RNAi to work in our fly species.
Impacts
Specific information on the molecular regulation of insect dormancy may yield novel strategies for the control of insect pests. Our results suggest that cell cycle regulation may play a significant role in the control of the developmental stasis observed during insect diapause.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
During year one of the granting period, we initiated studies designed to elucidate the molecular mechanism of diapause in two insect species. Within this grant, we are interested in defining the physiology of diapause termination as well as diapause programming. Our first aim was to define the regulation of the Proliferating Cell Nuclear Antigen (PCNA) gene during insect diapause. We previously reported that this gene was differentially expressed throughout the diapause program in the flesh fly, Sarcophaga crassipalpis. Semi-quantitative RT-PCR and quantitative Real-Time PCR were performed to determine the expression pattern of this gene product both during diapause and following diapause termination in the flesh fly. Results acquired from these experiments revealed that PCNA expression is basal during the entire period of pupal diapause, followed by a 15-fold transcriptional up-regulation within 12 hours after the termination of diapause. In situ hybridization using
laser scanning confocal microscopy verified these data and further defined the spatial distribution of this gene product in flesh fly brains following diapause termination. While PCNA transcriptional control appears to be important during this developmental stasis, we are also interested in defining the role of other cell cycle-associated genes in diapause. Using RT-PCR, we have generated clones of several other cell cycle regulatory genes from the flesh fly and the tobacco hornworm, Manduca sexta, a Lepidopteran insect that also overwinters in a pupal diapause. Portions of the Sarcophaga and Manduca PCNA, Cyclin E, and Dacapo genes were isolated and sequenced to verify their identities. Defining the transcriptional control of these genes should further our knowledge of cell cycle regulation in insect development, as well as insights into common strategies used by insects to control the developmental stasis observed during diapause. We have also launched experiments to define the
intracellular signaling pathways necessary for the initiation of adult development following diapause termination. Using immunoprecipitation, we have isolated four proteins that are phosphorylated on tyrosine residues between one and three hours after diapause termination. We are in the process of identifying these proteins via partial amino acid sequencing. We feel these results are quite exciting, as this represents kinase activity that precedes PTTH release after diapause termination. Our second area of study is the photosensitive stage that occurs during late embryonic/early larval life in the flesh fly. In order to understand the programming of diapause, which requires exposure to short blue light photoperiod, we cloned a portion of the blue light photoreceptor cryptochrome from S. crassipalpis. The clone was obtained using degenerative PCR oligonucleotide primers generated against Drosophila melanogaster cryptochrome. We are now attempting RNA interference (RNAi) to block the
production of functional cryptochrome. Results from these experiments should yield significant insight into the mechanism by which insects are entrained to enter diapause.
Impacts
Results from these experiments will significantly extend our knowledge base regarding the molecular mechanism(s) that control diapause programming and termination in insect species. With this information we will be able to target specific genes and/or gene products in order to abolish the dormancy period used by many agricultural pests to overcome the harsh conditions of winter.
Publications
- No publications reported this period
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