Steven J. SEYBOLD-1, David R. QUILICI-1, Julie A. TILLMAN-1,
Désirée VANDERWEL-2, David L. WOOD-3, and Gary J. BLOMQUIST-1
1-Department of Biochemistry, University of Nevada, Reno, NV 89557-0014
2-Department of Chemistry, University of Winnipeg, Winnipeg, Manitoba, Canada R3B 2E9
3-Department of Environmental Science, Policy, and Management, University of California,
Berkeley, CA 94720
The male California five-spined ips, Ips paraconfusus Lanier produces the myrcene-derived, acyclic monoterpene alcohols, ipsenol (2-methyl-6-methylene-7-octen-4-ol) (1) and ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) (2) as components of its aggregation pheromone attractant. Western North American populations of the male pine engraver beetle, Ips pini (Say), produce ipsdienol as an aggregation pheromone attractant and small quantities of ipsenol as an aggregation pheromone interruptant. These beetles tunnel and feed in the phioem tissue of pines, and the acyclic monoterpene myrcene is present in the xylem and phloem oleoresin that adjoins and pervades this tissue. Previous studies have shown that myrcene is a direct precursor to ipsdienol and, ultimately, ipsenol. In vivo radiolabeling studies reported here showed that male I. paraconfusus incorporated [1- 14C]acetate into ipsenol, ipsdienol, and amitinol (trans-2-methyl-6-methylene-3,7-octadien-2-ol) (3), while male I. pini incorporated [1- 14C]acetate into ipsdienol and amitinol. Females of these species produced neither labeled nor unlabeled pheromone components. The purified, radiolabeled monoterpene alcohols from males were identified by comparison of their HPLC and GC retention times with those of unlabeled standards. HPLC-purified fractions containing the individual radiolabeled components were analyzed by GC-MS and were shown to include only the pure alcohols. To further confirm that ipsdienol and ipsenol were radiolabeled, diastereomeric ester derivatives of the isolated alcohols were synthesized and analyzed by HPLC and GC-MS. Following derivatization of the radiolabeled alcohols, the HPLC analysis demonstrated expected shifts in retention times with conservation of naturally occurring enantiomeric composition. Additional radiotracer studies with (RS)-[5-3H] and (RS)-[2- 14C] mevalonolactone confirm that I. paraconfusus and I. pini utilize the isoprenoid pathway for this biosynthesis. In contrast to previous studies that have emphasized exogenous, host-derived precursors in pheromone biosynthesis, our results provide direct evidence for de novo biosynthesis of ipsenol, ipsdienol, and amitinol by bark beetles.