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The California five-spined ips, Ips paraconfusus Lanier, produces the myrcene-derived acyclic monoterpene alcohols ipsenol (2-methyl-6-methylene-7-octen-4-ol) and ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) as components of its aggregation pheromone. The pine engraver beetle, Ipspini (Say), produces only ipsdienol. Previous studies have shown that myrcene, a monoterpene in the pines colonized by these beetles, is a direct precursor to these pheromone components. In vivo radiolabeling studies reported hereshowed that male I. paraconjifsus incorporated [1_14C]acetate into ipsenol, ipsdienol, and amitinol (trans-2-methyl-6-methylene-3,7-octadien-2-ol), while male I. pini incorporated [1-_4C]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. HPLCpurified 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. After derivatization of the radiolabeled alcohols, the HPLC analysis demonstrated expected shifts in retention times with conservation of naturally occurring stereochemistry. The results provide direct evidence for de novo biosynthesis of ipsenol, ipsdienol, and amitinol by bark beetles. The aggregation pheromone components (4S)-(-)-ipsenol(2-methyl-6-methylene-7-octen-4-ol) (1) and (4S)-( + )-ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) (2) were isolated and identified from males of the California five-spined ips, Ips paraconfusus Lanier. These components, together with (1S,2S)-(+)-cis-verbenol (cis-4,6,6-trimethyl bicyclo[3.1.1]-hept-3-en-2-ol), were identified as the first coleopteran pheromone (1). The structural similarity of the acyclic monoterpene alcohols ipsenol and ipsdienol to myrcene (2, 3), a monoterpene present in the oleoresin of the principal host, ponderosa pine, Pinus ponderosa Laws., led to a deuterium labeling study with I. paraconfusus that unequivocally showed the in vivo transformation of myrcene to ipsenol and ipsdienol (4). A more recent labeling study demonstrated the conversion of myrcene to ipsdienol in the male pine engraver beetle, Ips pini (Say) (5). Recent reviews (6-9) emphasize the central role of plant-derived myrcene in the production of ipsenol and ipsdienol, including the hypothesis that myrcene from the host may be sequestered and bioaccumulated by males during early developmental stages for later use as adults (9 consistent with the general concept that monoterpenes sensu stricto are plant products (7,10,11).However, it has been recognized that alternative routes for Ips spp. pheromone biosynthesis may exist, such as de novo biosynthesis or utilization of other terpene hydrocarbo...

Evidence is presented for transcriptional regulation of de novo pheromone biosynthesis in Ips spp. bark beetles, but the comparative biochemical and molecular approach reveals a dichotomy between species in the pini and grandicollis subgeneric groups. Radiotracer studies with 14C-acetate demonstrate that feeding on host phloem stimulates biosynthesis in males of three Ips spp. However, treatment with juvenile hormone III (JH III) stimulates biosynthesis only in Ips pini. Thus, two species in the grandicollis subgeneric group (I. grandicollis and I. paraconfusus) appear to have a different mode of regulation related to JH III than does I. pini. Between 16 and 20 hr after feeding has commenced, pheromone production, as measured by accumulation in abdominal tissue, is stimulated about 150- (I. pini) and 350-times (I. paraconfusus) above the control level of 1-10 ng/male measured at 0 hr. Treatment with JH III results in accumulation in I. pini that is 3-4 times more than in phloem-fed males, whereas the identical treatment results in only weak accumulation in I. paraconfusus (45-times less than phloem-fed males). Comparative studies of gene expression and enzyme activity related to biosynthesis also support different modes of JH III-related regulation in I. pini and I. paraconfusus. In males of both species, feeding on host phloem results in increased transcript abundance and increased activity for the key de novo isoprenoid pathway enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). However, while JH III treatment results in comparable maximal increases in HMG-R transcript levels in both species (similar to feeding), the activity of HMG-R in crude extracts from JH III-treated male I. paraconfusus is low in comparison with male I. pini. Hypothetical explanations for the interspecific dichotomy in the regulation of pheromone biosynthesis include a second hormone or factor in grandicollis group species that functions either alone or with JH III; in both cases acting after HMG-R has been transcribed.