Progress 07/01/05 to 06/30/08
Outputs
OUTPUTS: We isolated Ico12D04, a cDNA encoding an oxidoreductase that converts ipsdienol to ipsdienone. This is a necessary intermediate step to produce the correct enantiomeric ipsdienol blend to act as a pheromone. We have characterized the mRNA distribution and are pursuing extensive biochemical functional assays of the recombinant enzyme following expression in a baculoviral system. Furthermore, an antibody for Ico12D04 has been produced, which greatly facilitates functional studies. Ico12D04 is a direct ortholog to Ipi12D04 The substrate and product profiles of both enzymes are being compared. 2. We worked to develop a reliable RNAi protocol for bark beetles by identifying siRNAs that knock down recombinant Ico12D04 production (determined by qRT-PCR and western blotting) in baculovirus-infected Sf9 cell cultures. Current work focuses on a reliable delivery method to live beetles. 3. We completed a careful survey of candidate normalizing genes for expression studies. This is a necessary tool for accurate gene expression analyses. 4. Two candidate GPPs cDNAs were identified and cloned from Ips confusus, and they are currently being characterized for their expression patterns and substrate/product profiles. PARTICIPANTS: Claus Tittiger: P.I., overall leadership on molecular biology Gary J. Blomquist: co-P.I., leadership on biochemical assays, beetle collection Matthew D. Ginzel: co-P.I., leadership on biochemical assays Rubi Figueroa-Teran: graduate student, Ico12D04 isolation and characterization, antibody characterization Jordan Naviaux: undergraduate student, Ico12D04 isolation and expression analysis Amy Griffith: undergraduate student, normalizing gene survey Michael Tanoue: undergraduate student, siRNA development and testing TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Ips confusus is an aggressive tree-killing beetle that attack both Colorado and single leaf pinyon pines. In fact, the largest outbreak of I. confusus ever recorded is affecting drought-stressed pinyons throughout Nevada and other parts of the Southwest. The long term goal of this project is to develop effective pest management tactics targeting the chemically-mediated mating system of the beetles. Populations may be managed, at least at a local level, by disrupting pheromone biosynthesis. 1. Determining that CYP9T1 does not produce the pheromonal blend of ipsdienol in Ips confusus confirmed the requirement for downstream enzymes to "tune" the CYP9T1 product. It has shifted our focus onto the oxidoreductase (Ico12D04) that almost certainly participates in this "tuning." Comparing the structures and activities of the oxidoreductases between Ips confusus and Ips pini will shed light on how pheromone systems evolve. Future control strategies designed based on pheromone systems will benefit from this insight. 2. Whereas we have been relying on cytoplasmic actin as a normalizing gene in previous studies, we now use that gene along with CYP27B1 and the qBASE algorithm for all normalizing studies. These are the two most stably-transcribed genes we have identified. 3. We determined that delivering siRNAs by allowing the beetles to feed through impregnated Kimwipes into pine phloem was not a reliable method because of difficulty controlling delivery. We therefore are now focusing on a more direct feeding delivery method.
Publications
- M. D. Ginzel, J. C. Bearfield, C. I. Keeling, C. C. McCormack, G. J. Blomquist and C. Tittiger, 2007. Antennally-mediated negative-feedback regulation of pheromone production in the pine engraver beetle, Ips pini. Naturwissenschaften. 91:61-64.
- P. Sandstrom, M.D. Ginzel, J.C. Bearfield, W.H. Welch, G.J. Blomquist, and C. Tittiger, 2008. Myrcene hydroxylases do not determine enantiomeric composition of pheromonal ipsdienol in Ips spp. J. Chem. Ecol. 34:1584-1592.
- International Society for Chemical Ecology Annual Meeting, Jena, Germany (July 2007). Entomological Society of America Annual Meeting, San Diego, CA (Dec. 2007). Entomological Society of America Annual Meeting, Reno, NV (Nov. 2007)
- Tehcnology transfer: A provisional patent for Ipi12D04 was filed: C. Tittiger, R. Figueroa-Teran, G.J. Blomquist (2008). Method for producing monoterpene and monoterpenoid compounds and use thereof. U.S. Provisional Patent, filed Nov 17, 2008. UNR09-003.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: Research has concentrated on pheromone biosynthetic enzymes, particularly those near the end of the ipsdienol-biosynthetic pathway. 1. We have isolated Ico12D04, a cDNA encoding an oxidoreductase that converts ipsdienol to ipsdienone. This is a necessary intermediate step to produce the correct enantiomeric ipsdienol blend to act as a pheromone. We have characterized the mRNA distribution and completed preliminary functional assays of the recombinant enzyme following expression in a baculoviral system. Furthermore, an antibody for Ico12D04 has been produced, which greatly facilitates functional studies. 2. We are progressing toward a reliable RNAi protocol for bark beetles by identifying siRNAs that knockdown recombinant Ico12D04 production (determined by qRT-PCR and western blotting) in baculovirus-infected Sf9 cell cultures. Current work focuses on a reliable method to deliver these siRNAs to live beetles. 3. We have completed a careful survey of candidate
normalizing genes for expression studies. This is a necessary tool for accurate gene expression analyses. 4. Two candidate GPPs cDNAs have been identified from Ips confusus, and they are currently being characterized for their expression patterns and substrate/product profiles. Events: Results from this research have been presented at the following conferences: 1. International Society for Chemical Ecology Annual Meeting, Jena, Germany (July 2007) 2. Entomological Society of America Annual Meeting, San Diego, CA (Dec. 2007)
PARTICIPANTS: Claus Tittiger: P.I., overall leadership on molecular biology. Gary J. Blomquist: co-P.I., leadership on biochemical assays, beetle collection. Matthew D. Ginzel : co-P.I., leadership on biochemical assays. Rubi Figueroa-Teran (grad student): Graduate student, Ico12D04 isolation and characterization, antibody characterization. Jordan Naviaux (undergraduate student): Ico12D04 isolation and expression analysis. Amy Griffith (undergraduate student): normalizing gene survey. Michael Tanoue (undergraduate student): siRNA development and testing. Desiree Vanderwel (U. Winnipeg, Canada): Collaborator, producing radio-labeled substrates necessary for 12D04 substrate/product profile determination. Training and professional development: Funds from this grant contributed to training of at least four undergraduate students and a graduate students in molecular biology and biochemistry.
Impacts
Ips confusus is an aggressive tree-killing beetle that attack both Colorado and single leaf pinyon pines. In fact, the largest outbreak of I. confusus ever recorded is affecting drought-stressed pinyons throughout Nevada and other parts of the Southwest. The long term goal of this project is to develop effective pest management tactics targeting the chemically-mediated mating system of the beetles. Populations may be managed, at least at a local level, by disrupting pheromone biosynthesis. 1. Determining that CYP9T1 does not produce the pheromonal blend of ipsdienol in Ips confusus confirmed the requirement for downstream enzymes to "tune" the CYP9T1 product. It has shifted our focus onto the oxidoreductase (Ico12D04) that almost certainly participates in this "tuning." Comparing the structures and activities of the oxidoreductases between Ips confusus and Ips pini will shed light on how pheromone systems evolve. Future control strategies designed based on pheromone
systems will benefit from this insight. 2. Whereas we have been relying on cytoplasmic actin as a normalizing gene in previous studies, we now use that gene along with CYP27B1 and the qBASE algorithm for all normalizing studies. These are the two most stably-transcribed genes we have identified. 3. RNAi has potential as a delivery mode for target-specific insecticides. Through using siRNAs it may be possible to knock down important genes related to a beetle's fitness, survival and reproduction. Because siRNAs could be developed against bark beetle-specific genes, the resulting control method would be species-specific, safe, and effective.
Publications
- Ginzel,M. D., Bearfield, J. C., Keeling, C. I. McCormack, C. C. Blomquist, G. J., and Tittiger, C. 2007. Antennally-mediated negative-feedback regulation of pheromone production in the pine engraver beetle, Ips pini. Naturwissenschaften. 91:61-64.
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Progress 01/01/06 to 12/31/06
Outputs
We previously demonstrated that feeding by male Ips confusus stimulates the release of a brain factor that works in concert with juvenile hormone (JH) III to stimulate pheromone production. We have isolated cDNAs for major pheromone-biosynthetic enzymes: HMG-S, HMG-R, GPPS, and IcoCYP9T1 and further characterized the effects of these two regulatory signals on their expression (with the exception of CYP9T1), including the time course of induction of their corresponding genes. We found that JH III seems to stimulate gene expression, while the additional brain factor is required for enzyme activity. Isolation of CYP9T1 is especially exciting because it is the enzyme that converts myrcene to ipsdienol (a major pheromone component). CYP9T1 produces mostly (-)-ipsdienol, even though the pheromone blend contains mostly (+)-ipsdienol. The ortholog in Ips pini (CYP9T2) has 94% sequence identity and the same product profile. These experiments show that final pheromone blends
are tuned by enzymes acting downstream of the production of the components. In this grant, we propose to design short anti-sense (interfering) RNAs (siRNA) to a number of mevalonate pathway genes, starting with HMG-R, which catalyzes the first committed, and thus most highly regulated, step in the mevalonate pathway and is a good target to knock-down pheromone production. We are working to develop and optimize the means to deliver siRNA to adult beetles. We have observed a knock-down effect of siRNAs specific to HMG-R in male I. pini, but need to develop a better delivery system. Thus, we are working to develop a feeding-based delivery strategy, rather than injections.
Impacts
Ips confusus is an aggressive tree-killing beetle that attack both Colorado and single leaf pinyon pines. In fact, the largest outbreak of I. confusus ever recorded is affecting drought-stressed pinyons throughout Nevada and other parts of the Southwest. The long term goal of this project is to develop effective pest management tactics targeting the chemically-mediated mating system of the beetles. Populations may be managed, at least at a local level, by disrupting pheromone biosynthesis. Analysis of CYP9T1 activity has opened our eyes to how pheromone blends are regulated. This information is crucial for understanding pheromone production, and ultimately in developing pheromone-based control strategies. RNAi has potential as a delivery mode for target-specific insecticides. Through using siRNAs it may be possible to knock down important genes related to a beetle's fitness, survival and reproduction. Because siRNAs could be developed against bark beetle-specific
genes, the resulting control method would be species-specific, safe, and effective.
Publications
- No publications reported this period
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Progress 07/01/05 to 12/31/05
Outputs
In the short amount of time since this grant was funded, we have made progress on two of the three stated objectives. We demonstrated that feeding by male Ips confusus stimulates the release of a brain factor that works in concert with juvenile hormone (JH) III to stimulate pheromone production. Head extracts from fed and unfed males were injected into JH III treated and untreated males that had not fed. We found the major pheromone component, ipsenol, was produced only by those males that had been treated with JH III. Interestingly, JHIII application alone is sufficient to induce pheromone production in male I. pini, a congener of I. confusus. In a number of other insect taxa a brain hormone is also necessary for production and release of pheromones. For example, in the lepidopterans Helothis zea and Bombyx mori, hormones called pheromone biosynthesis activating neuropeptides are responsible for pheromone synthesis and release. These 33 or 34 amino acid peptides
alter the activity of enzymes involved in pheromone biosynthesis. With this in mind, we aligned amino acid sequences of PBANs from 16 species representing a variety of taxa and designed degenerate primers based on their homology. We are currently optimizing PCR conditions for these primers. Bark beetles, including I. confusus, produce pheromone components de novo via the mevalonate pathway. In this grant, we propose to design short anti-sense (interfering) RNAs (siRNA) to a number of mevalonate pathway genes. This RNAi technique can trigger a natural cellular response resulting in the attenuation or complete loss of mRNA for that gene. As a first step, we have used primers specific to I. pini to isolate a 100 amino acid fragment of 3-hydroxy-3-methylglutary-CoA reductase (HMG-R) in I. confusus. This enzyme catalyzes the first committed, and thus most highly regulated, step in the mevalonate pathway and is a good target to knock-down pheromone production. Moreover, substantial progress
has been made in developing and optimizing the means to deliver siRNA to adult beetles. In fact, using quantitative real-time PCR, we were able to confirm a knock-down effect of siRNAs specific to HMG-R in male I. pini.
Impacts
Ips confusus is an aggressive tree-killing beetle that attack both Colorado and single leaf pinyon pines. In fact, the largest outbreak of I. confusus ever recorded is affecting drought-stressed pinyons throughout Nevada and other parts of the Southwest. Over the past two years, I. confusus has killed over 3.1 million pinions in Nevada alone. We hope research on pheromone biogenesis will lead to the development of new control methods-enhancing the health, quality, and productivity of Nevada's pinyon pines. The long term goal of this project is to develop effective pest management tactics targeting the chemically-mediated mating system of the beetles. Populations may be managed, at least at a local level, by disrupting pheromone biosynthesis. Moreover, RNAi has potential as a delivery mode for target-specific insecticides. Through using siRNAs it may be possible to knock down important genes related to a beetle's fitness, survival and reproduction. Because siRNAs could
be developed against bark beetle-specific genes, the resulting control method would be species-specific, safe, and effective.
Publications
- No publications reported this period
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