Progress 10/01/01 to 09/30/04
Outputs
Objective 1: Using the mRNA for HMG-CoA reductase as a marker of pheromone biosynthesis, the site of frontalin production was determined in Dendroctonus jeffreyi using in situ hybridization. These results were confirmed with biochemical assays. Electron micrograph studies revealed dramatic changes in the sub-cellular organization of pheromone-biosynthetic cells accompany pheromone production. Similar studies could not definitively identify the site of pheromone production in Anthonomus grandis, though biochemical and molecular data strongly support our hypothesis that pheromones in that species are produced in digestive tissues. Objective 2: Based on the precedent established for monoterpenoid phermone production in bark beetles, we isolated and characterized HMG-CoA reductase (HMG-R) from the cotton boll weevil (A. grandis). There is a generally increasing trend in HMG-R mRNA in males over time, and expression levels are higher in midguts than in fat bodies. These
data correlate with pheromone biosynthesis, however, neither feeding nor topical JH III treatment significantly change HMG-R expression levels. While this is in contrast to the situation observed in bark beetles, it is consistent with observations that male boll weevil JH III levels are constitutively low. These data confirm that signals other than JH III are regulating boll weevil pheromone production. Objective 3: HMG-R and HMG-S genes from Dendroctonus jeffreyi were isolated and molecularly characterized. Expression patterns in response to JH III were thoroughly investigated: JH III stimulates HMG-R and HMG-S gene expression in males, but not females, consistent with the genes' role in monoterpenoid pheromone production. Developmental studies showed that the HMG-R gene becomes competent to respond to JH III while teneral adults tunnel out from the brood tree. Other: Characterization of fatty acyl-CoA desaturase genes from the house fly and crickets was finalized. A functional
genomics study was performed on pheromone-biosynthetic tissues from Ips pini. An EST database (1671 clones, 574 tentative unique genes) was assembled, and microarrays were fabricated. The ESTs revealed a new family of apparently scolytid-specific genes that may be involved in phloem detoxification. Microarray experiments revealed coordinate regulation of the mevalonate pathway in both male and female midguts. Late steps in the pathway are regulated differently between the sexes, and basal expression levels are much higher in males compared to females. This information provides a more complete picture of how genes respond to JH for pheromone biosynthesis in these cells. The functional genomics data also revealed two new interesting genes: one encodes a cytochrome P450 that is strongly implicated in the conversion of myrcene to ipsdienol, and the other encodes an uncharacterized protein, conserved in a variety of insects, that may be involved in a signal transduction pathway and thus
may have a role in JH signaling. Other cytochromes P450 have been isolated and are being tested for possible roles in phloem detoxification.
Impacts
The research strengthened the new paradigm that the midgut of some beetles, particularly the Scolytidae and Curculionidae, is a pheromone-biosynthetic tissue. This should impact thinking about future control strategies involving inhibiting pheromone biosynthesis- i.e. feeding may be an attractive delivery mode since it would deliver a pesticide directly to the target tissue. Characterization of HMG-S and HMG-R genes in D. jeffreyi provided the first molecular evidence that frontalin is produced de novo via the mevalonate pathway and served to focus biochemical experiments in that area. The ESTs and associated microarrays are the first examples of functional genomics studies in bark beetles and represent pioneering efforts to use functional genomics to study pheromone biosynthesis and JH-regulation in a non-model organism. Information from these experiments invokes new thinking about how bark beetle cells are able to synthesize massive amounts of pheromone components.
They have also revealed several new genes and initiated molecular studies into how bark beetles are able to thrive in the toxic environment of the tree.
Publications
- Eigenheer AL, Young S, Blomquist GJ, Borgeson CE, Tillman JA, Tittiger C. 2002. Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences. Insect Molec. Biol. 11: 533-42.
- Hall GM, Tittiger C, Andrews GL, Mastick GS, Kuenzli M, et al. 2002. Midgut tissue of male pine engraver, Ips pini, synthesizes monoterpenoid pheromone component ipsdienol de novo. Naturwissenschaften 89: 79-83.
- Hall GM, Tittiger C, Blomquist GJ, Andrews GL, Mastick GS, et al. 2002. Male jeffrey pine beetle, Dendroctonus jeffreyi, synthesizes the pheromone component frontalin in anterior midgut tissue. Insect Biochem. Molec. Biol. 32: 1525-32.
- Nardi J, Young A, Ujhelyi E, Tittiger C, Lehane M, Blomquist GJ. 2002. Specialization of midgut cells for synthesis of male isoprenoid pheromone components in two scolytid beetles, Dendroctonus jeffreyi and Ips pini. Tissue and Cell 34: 221-31.
- Riddervold MH, Tittiger C, Blomquist GJ, Borgeson CE. 2002. Biochemical and molecular characterizaton of house cricket (Acheta domesticus, Orthoptera: Gryllidae) Delta9 desaturase. Insect Biochem. Molec. Biol. 32: 1731-40.
- Eigenheer, A.L., Keeling, C.I., Young, S. and Tittiger, C. 2003. Comparison of gene representation in midguts from two phytophagous insects, Bombyx mori and Ips pini, using expressed sequence tags. Gene 316C, 127-136.
- Keeling CI, Young S, Tittiger C. 2005. Juvenile hormone-regulated gene expression in the anterior midgut of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae). In preparation.
- Taban AH, J. F, Blake J, Awano A, Tittiger C, Blomquist G. 2005. HMG-CoA reductase and site of pheromone biosynthesis in the cotton boll weevil, Anthonomus grandis. Insect Biochem. Molec. Biol.
- Seybold, S.J. and Tittiger, C. 2003. Biochemistry and molecular biology of de novo isoprenoid pheromone production in the Scolytidae. Ann Rev. Entomol. 48: 425-453.
- C. Tittiger 2003. Molecular biology of bark beetle pheromone production and endocrine regulation. In Insect Pheromone Biochemistry and Molecular Biology, G.J. Blomquist and R. Vogt, eds. Elsevier.
- Tittiger, C., Barkawi, L.S., Bengoa, C.S., Blomquist, G.J. and Seybold, S.J. 2003. Structure and juvenile hormone-mediated regulation of the HMG-CoA reductase gene from the Jeffrey pine beetle, Dendroctonus jeffreyi. Molec. Cell. Endocrinol. 199, 11-21.
- Blomquist GJ, Jurenka RA, Schal C, Tittiger C. 2004. Biochemistry and molecular biology of pheromone production. In Comprehensive Insect Physiology, Biochemistry, Pharmacology, and Molecular Biology, ed. LI Gilbert, K Iatrou, SS Gill: Elsevier
- Keeling CI, Blomquist GJ, Tittiger C. 2004. Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae). Naturwissenschaften 91: 324-8.
- Tillman JA, Lu F, Staehle L, Donaldson Z, Dwinell SC, et al. 2004. Juvenile hormone regulates de novo isoprenoid aggregation pheromone biosynthesis in pine bark beetles, Ips spp. (Coleoptera: Scolytidae), through transcriptional control of HMG-CoA reductase. J. Chem. Ecol. 30: 2459-94.
- Tittiger C. 2004. Functional Genomics and Insect Chemical Ecology. J. Chem. Ecol. 30: 2335-58.
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Progress 01/01/03 to 12/31/03
Outputs
Objective 1: Confirm the anatomical location of pheromone biosynthesis in A. grandis and D. jeffreyi. Prior to the current reporting period, biochemical studies of Ips pini and D. jeffreyi bark beetles confirmed that the midgut is the site of pheromone biosynthesis. Electron microscopy revealed dramatic changes in sub-cellular organization of pheromone-biosynthetic cells accompany pheromone production. In A. grandis, northern blots show HMG-R gene expression is higher in midguts compared to fat bodies, which correlates with radio-tracer studies showing midguts incorporate metabolic precursors into pheromone components more readily than fat bodies. These data strongly support our hypothesis that digestive tissues synthesize pheromone components in A. grandis. Objective 2: Determine the regulation of A. grandis pheromone production. HMG-R mRNA levels generally increase with the age of male A. grandis in a pattern that correlates with aggregation pheromone biosynthesis.
However, neither feeding nor topical JH III treatments significantly changed HMG-R mRNA levels. The lack of response to JH III is not surprising given reports in the literature that adult A. grandis JH titres are consistently low. These data confirm that signals other than feeding and JH III regulate boll weevil aggregation pheromone production. Objective 3: Characterize A. grandis and D. jeffreyi HMG-R and HMG-S genes. HMG-S gene studies in D. jeffreyi were completed and published before the current reporting period. The D. jeffreyi HMG-R gene was fully isolated and sequenced. Its intron/exon organization suggests functional roles for introns in the membrane anchor coding region. The regulation of HMG-R expression by JH III was throroughly investigated: JH III stimulates HMG-R gene expression in males, but not females, consistent with the gene's role in monoterpenoid pheromone production. Developmental studies suggest that the gene becomes competent to respond to JH III while the
teneral adult tunnels out from the brood tree. A full-length HMG-R cDNA was isolated and characterized from A. grandis. A functional genomics project was initiated to identify components of the pheromone biosynthetic and endocrine regulatory apparati in I. pini. To this end, we made an expressed sequence tag (EST) database containined 574 genes expressed in male midguts and assoicated microarrays. These are being screened to identify JH-responsive and pheromone biosynthetic genes.
Impacts
This work resolved a long-standing debate about the site of pheromone biosynthesis in bark beetles and enabled a functional genomics project in Ips pini. The functional genomics results should yield new molecular targets that may be used to control pest bark beetles.
Publications
- Seybold, S.J. and Tittiger, C. 2003. Biochemistry and molecular biology of de novo isoprenoid pheromone production in the scolytidae. Annu. Rev. Entomol. 48, 425-453.
- Tittiger, C., Barkawi, L.S., Bengoa, C.S., Blomquist, G.J. and Seybold, S.J. 2003. Structure and juvenile hormone-mediated regulation of the HMG-CoA reductase gene from the Jeffrey pine beetle, Dendroctonus jeffreyi. Molec. Cell. Endocrinol. 199, 11-21.
- Tittiger, C.: Molecular biology of bark beetle pheromone production and endocrine regulation. In: Blomquist, G.J., Vogt, R. (Ed.), Insect Pheromone Biochemistry and Molecular Biology. Elsevier, San Diego, 2003.
- Eigenheer, A.L., Keeling, C.I., Young, S. and Tittiger, C. 2003. Comparison of gene representation in midguts from two phytophagous insects, Bombyx mori and Ips pini, using expressed sequence tags. Gene 316C, 127-136.
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Progress 01/01/02 to 12/31/02
Outputs
An expressed sequence tag database of pheromone-biosynthetic Ips pini midguts was completed. The database contains 1671 clones, representing 574 unique loci. These include six mevalonate pathway genes, juvenile hormone-degrading enzymes (JH esterase and JH epoxide hydrolase), and numerous clones with unknown function. When compared to a similar EST dataset from the larval midguts of the silkworm, Bombyx mori, the distribution of functional classes shows that genes important for general metabolism and energy production are more highly represented in the bark beetle guts compared to the silkworm. This is consistent with the dual roles of metabolism and pheromone production that bark beetle midguts must perform. Full-length cDNAs have been isolated for boll weevil (Anthonomus grandis) farnesyl diphosphate synthase, and for Jeffrey pine beetle (Dendroctonus jeffreyi) farnesyl diphosphate synthase, thiolase, and isopentenyl diphosphate isomerase. Comparitive expression
studies of these genes with respect to pheromone biosynthesis are in progress.
Impacts
The EST project in Ips pini has revealed several new unknown clones, and confirmed the extraordinary metabolic state of the midgut. This information may lead to new pest control measures. A comparison of the expression patterns of different mevalonate pathway genes in boll weevils and bark beetles should be helpful to distinguish how pheromones are synthesized.
Publications
- Eigenheer, A.L., Young, S., Blomquist, G.J., Borgeson, C.E., Tillman, J.A., Tittiger, C. 2002. Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences. Insect Molecular Bilogy 11(5): 533-542.
- Tittiger,C. Barkawi, L.S., Bengoa, C.S., Blomquist, G.J., Seybold, S.J. 2003. Structure and juvenile hormone-mediated regulation of the HMG-CoA reductase gene from the Jeffrey pine beetle, Dendroctonus jeffreyi. Mol. Cel. Endocrin. In press.
- Seybold, S.J. and Tittiger, C. 2003. Biochemistry and molecular biology of de novo isoprenoid pheromone production in the Scolytidae. Ann Rev. Entomol. 48: 425-453.
- C. Tittiger 2003. Molecular biology of bark beetle pheromone production and endocrine regulation. In Insect Pheromone Biochemistry and Molecular Biology, G.J. Blomquist and R. Vogt, eds. In press.
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Progress 01/01/01 to 12/31/01
Outputs
Approximately 1.8 kb of upstream DNA flanking the HMG-CoA reductase gene from the Jeffrey pine beetle (Dendroctonus jeffreyi) was isolated using TAIL-PCR. The DNA likely has promoter elements that make the gene sensitive to juvenile hormone, and will thus be useful for studying how the hormone regulates pheromone biosynthesis. The expression pattern of the HMG-R gene was also investigated in immature adult males, providing evidence that the ability for the gene to respond to the hormone (i.e. competence) is acquired rapidly during maturation, and that newly emerged beetles have two distinct phenotypes: those strongly expressing HMG-R and those that are not. An expressed sequence tag (EST) database representing 1321 different clones from pheromone-producing tissue from Ips pini was generated with the help of the Nevada Genomics Center. This effort has yielded numerous candidate clones for important biosynthetic and regulatory genes,including juvenile hormone-degrading
enzymes that are hypothesized to have great importance inregulating hormone levels. A full length cDNA for HMG-CoA reductase from the boll weevil (Anthonomus grandis) was isolated and sequenced. Expression of this gene correlates loosely with pheromone release, raising the possibility that pheromone components are synthesized through modification of cotton plant precursor molecules. A full length cDNA for farnesyl diphosphate synthase (FPPS) was also isolated and characterized through expression studies and molecular modeling.
Impacts
An understanding of the anatomical site of pheromone synthesis should impact the design of future pest control strategies. For example, if the precedent that pheromones are synthesized in midgut tissues, set in bark beetles, holds true in boll weevils, then is may be possible to deliver specific inhibitors by feeding rather than spraying. An understanding of how boll weevils and bark beetles regulate pheromone biosynthesis through the use of hormones will make additional targets for disruption available. These data will be useful in guiding the development of more effective, targeted, and safe pesticides to control these significant pests.
Publications
- Hall, G., Tittiger C.,. Andrews G.L,. Mastick G.S,. Kuenzli M,. Luo X, Seybold S.J. and G.J. Blomquist (2002). Male pine engraver midgut tissue synthesizes monoterpenoid pheromone de novo. Naturwissenschaften, in press.
- Nardi, J.B.,. Young A.G, Tittiger C., Lehane M.J., and. Blomquist, G.J (2002). Specialization of midgut cells for synthesis of male isoprenoid pheromone components in two scolytid beetles, Dendroctonus jeffreyi and Ips pini. Tissue and Cell, in press.
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