Byers, J.A. and Birgersson, G. 1990. Pheromone production in a bark beetle independent of myrcene precursor in host pine species. Naturwissenschaften 77:385-387. pdf

The two large pines in background are digger pine, Pinus sabiniana (at left) and ponderosa pine, Pinus ponderosa (at right).
The California five-spined engraver, Ips paraconfusus (Coleoptera: Scolytidae), is an important pest of young pine forests in California and Oregon, where nearly all pine species within the range of this bark beetle are attacked [1]. The aggregation pheromone produced by males has been identified as a synergistic blend of three components, (S)-(-)-ipsenol, (S)- (+)-ipsdienol, and (4S)-cis-verbenol [2]. Ipsenol and ipsdienol accumulate only in males exposed to vapors of the host tree monoterpene, myrcene, in a logarithmic relationship [3]. Recently emerged control males and females do not contain volatile terpene compounds, neither pheromone components nor myrcene, while only myrcene is present in vapor-exposed females [3]. Hendry et al. [4] used deuterium-labelled myrcene to prove that myrcene could be converted by hydroxylation to ipsenol and ipsdienol in males exposed to vapors. Another host tree monoterpene, (-)-alpha-pinene, in the vapor phase is converted to cis-verbenol in both sexes [5, 6].

A paradigm has been established that I. paraconfusus, and probably most other bark beetles of the genus Ips, use myrcene and alpha-pinene in their host tree as the precursors to the aggregation pheromone components, ipsenol/ipsdienol and cis-verbenol, respectively [3-8]. Host selection and host suitability may then depend, in part, on the concentration of myrcene and alpha-pinene in the tree [9]. Pine trees exhibit wide variation within and between species in their composition of myrcene and alpha-pinene, among other monoterpenes [10, 11]. Thus it has also been hypothesized that tree genotypes could have evolved to have lower titres of aggregation pheromone precursors as part of a resistance mechanism to bark beetles [8].

We compared the relative attractiveness of five pine host species, ponderosa (Pinus ponderosa), sugar (P. lambertiana), Jeffrey (P. jeffreyi), digger (P. sabiniana), and lodgepole (P. contorta), that were infested with I. paraconfusus in order to detect possible differences in pheromone release and host suitability.
Pine cone on left is from Jeffrey pine while that on right is from ponderosa pine.
Beetles were reared from naturally-infested ponderosa pine collected from the Sierra National Forest, California. Fifty males were introduced (18:00 Aug. 29, 1985) to holes drilled in logs of each of the five host pines cut two days previously. These logs were wrapped with window screen and placed in sticky traps. A trap consisted of 6-mm mesh screen cylinders (19 cm diam. x 30.5 cm high) coated with Stikem Special R at 1.2 m height. Traps were separated 10 m apart in a line (Sierra National Forest, California).

Collections of flying beetles at each of the five infested logs were similar except for an approximate doubling of catch on the Jeffrey pine log (Fig. 1 A).

Fig. 1. (A) Catch of Ips paraconfusus attracted to logs (28 cm x 15 cm diam.) of ponderosa pine (PP), sugar pine (SP), Jeffrey pine (JP), digger pine (DP), and lodgepole pine (LP) each infested with 50 male I. paraconfusus (Aug. 31 - Sept. 2, 1985). The catch on Jeffrey pine (asterisk) was significantly more than on either PP, SP, or DP, but not LP (arcsin transformations, t-tests, P< 0.01, n=4).
(B) Amounts of pheromone components ipsenol, ipsdienol and (4S)-cis-verbenol in the hindguts of male Ips paraconfusus after feeding in either PP, SP, JP, DP or LP (above) for 5 days (Aug. 29 - Sept. 3, 1985). Quantification was by GC-MS on a Finnigan model 4021 using a column of fused silica (0.15 mm i.d. x 25 m) coated with SuperoxR FA (Alltech, TPA-treated PEG, df=0.3 um) on a temperature program of 50o for 4 min, rising to 200o at 8o/min and isothermal for 10 min and helium carrier gas at 25 cm/s. Limit of detection was 0.03 ng/male.
(C) Amounts of alpha-pinene and myrcene in phloem (dry weight) from logs fed on by Ips paraconfusus males (Sept. 3, 1985). Phloem extracts were analyzed by GC using a fused silica column (0.2 mm i.d. x 12.5 m) coated with SER-54 CL (General Electric, 1% vinyl- , 5% phenyl-, 94% methylpolysiloxane) on a temperature program of 60oC for 3 min, rising to 220oC at 5o/min and isothermal for 15 min. Nitrogen, 20 cm/s, was used as carrier gas. Identities of the monoterpene hydrocarbons were confirmed using GC-MS as in (B) above. Vertical lines atop bars (B, n=5; C, n=3) represent the S.E.M.

In all cases, attraction rates were significant since one to two or more females could have joined a male in his nuptial chamber if not intercepted by the traps, a natural pairing ratio in I. paraconfusus [12]. Another measure of the strength of a pheromone signal is the sex ratio of catch; a higher female-biased ratio in I. paraconfusus indicates a higher release of pheromone, because high release rates cause inhibition of male response [12]. The collected sex ratios were similar to previous reports [12], though the sex ratio on Jeffrey pine ( : = 1:15.6) was significantly higher than on the other pine species (Fig. 1 A), again indicating that a somewhat more potent attractant was released. Five days after introducing the males, the logs were dissected and no significant differences were noted in survival or in general appearance of nuptial chambers (43-47 per log).

To determine the quantities of the pheromone components ipsenol, ipsdienol and cis- verbenol in the feeding males, they were removed from nuptial chambers and their hindguts extracted in groups of 8 in 150 ul diethyl ether with 10 ng heptyl acetate per ul as an internal quantification standard. Monoterpenes, including myrcene and alpha-pinene, were extracted similarly from three samples of uninfested phloem (15-25 mg dry weight) from each of the infested logs using 250 ul ether per sample, as well as from oleoresin (Table 1).
Table 1. Amounts of alpha-pinene and myrcene in phloem samples (g dry weight, n=3) from five species of pine fed on by Ips paraconfusus (Sept. 3, 1985) and in oleoresina (Oct. 17, 1986). The major monoterpene in ponderosa and Jeffrey oleoresin was 3-carene (498.5 ug/ul and 16.4 ug/ul, respectively), while alpha-pinene predominated in digger, and lodgepole resin was mostly limonene (723 ug/ul). Total monoterpene percentage (including alpha- and beta-pinene, myrcene, 3-carene and limonene) of oleoresin was 83.5% for ponderosa, 89.9% for lodgepole, 2.9% for Jeffrey and 0.08% for digger, consistent with n-heptane as the major constituent of oleoresin in the latter two species [11].
Pine species Amount ( ug per g phloem or ul resin )
Ponderosa phloem < 1.3 - 16.41.6 - 6.6
oleoresina34.9 - 48.8 94.9 - 139.0
Sugar phloem3.8 - 7.92.4 - 2.6
Jeffrey phloem152 - 44515.8 - 52.6
Digger phloem< 0.01< 0.01
oleoresin0.68< 0.01
Lodgepole phloem35.0 - 76.318.6 - 39.0
aFour samples from cardinal directions of one tree.
Pheromone components and ipsenone in the males, and monoterpene hydrocarbons from the host were quantified by gas chromatography and mass spectrometry (GC-MS) using fused silica capillary columns (Fig. 1) and interpreted with respect to synthetic chemical standards (from Borregaard, Sarpsborg, Norway, and Aldrich chemical company, Milwaukee, Wisconsin). Ipsenone was prepared by oxidation of ipsenol in Jones reagent [13].

The hindguts contained only a few major components, with ipsenol and ipsdienol dominating. Ipsenone, the ketone corresponding to ipsenol, was observed for the first time in I. paraconfusus, in males feeding in all pine species at 112± 30 (ng/male± SD, n=5). Ipsenone, rather than ipsdienone which has not been found, may account for the observed loss of deuterium from carbon 4 during the conversion of ipsdienol to ipsenol [7]. The generally similar attraction to each of the pine species agrees with the similar amounts of the pheromone components, ipsenol and ipsdienol, found in the hindguts of the feeding males (Fig. 1 B). The increased catch on Jeffrey, and to a lesser extent on lodgepole, can be explained by the higher amounts of cis-verbenol, the third pheromone component (Fig. 1 B). Although cis- verbenol was undetected by GC-MS (< 0.5 ng/male) in males feeding in the other pines, it was presumably released in sufficient quantities to be synergistically active with ipsenol and ipsdienol [2].

Unless enzyme saturation occurs even at the lowest concentrations of precursor found in nature, the paradigm predicts there should be a positive relationship between amounts of precursors in the host and pheromone products in the beetle. There appears to be a positive correspondence between higher levels of alpha-pinene in Jeffrey and lodgepole pines and levels of cis-verbenol in males from these species (Figs. 1 B-C). However, there was no significant relationship between the widely varying amounts of myrcene in the host pines and the uniform amounts of ipsenol and ipsdienol in the males (slopes were not different from 0, t-test; Figs. 1 B- C). The absence of myrcene in digger pine is in agreement with earlier reports by Mirov [11] that show the oleoresin contains 95% n-heptane, and no mention of monoterpenes. There was a moderate variation within a pine species of alpha-pinene and myrcene that was not as large as between species (ranges in table) and was probably due to uneven distributions of resin pockets among the rather small sample units (15-25 mg dry weight). However, the sample unit was equivalent to about 80% of a beetle's "nuptial chamber" and thus indicates that individuals in the same or different trees could ingest large differences in monoterpene hydrocarbons such as myrcene [6].

Top kill of ponderosa pines by <I>Ips paraconfusus</I> in California
Top kill of ponderosa pines by Ips paraconfusus in California
The question arises, is there enough myrcene in the beetle's diet (host phloem) or in oleoresin to account for the quantities of ipsenol and ipsdienol found in the hindguts? The vapor concentration of myrcene in a nuptial chamber of ponderosa pine (2.8 x 10-8 g/ml) is expected to account for only 2% at most of the ipsenol in feeding males [6]. The absence of myrcene in digger pine phloem makes it unlikely that this compound could serve as a precursor. Allowing that a trace of myrcene was present in the oleoresin of digger pine (at most 0.01 ug/ul, our quantification limit), and assuming very conservatively that this was converted completely to the components and no components were released, a beetle would need to eat at least 80 ul or 8 times its weight in oleoresin, and even higher amounts to account for releases of pheromone during the feeding period. This appears to be virtually impossible since the males' guts were observed to contain phloem, and oleoresin is toxic to bark beetles (I. paraconfusus and Dendroctonus brevicomis) [6, 14].

From our results it is apparent that all five pine species are about equally suitable as hosts, at least in terms of adult survival, nuptial chamber construction, pheromone production and attraction. The production of similar amounts of ipsenol and ipsdienol in all pine species regardless of myrcene titre, as well as the inadequate supply of myrcene in digger pine, indicate that males may utilize precursors other than myrcene predominantly. This implies that insect-plant coevolution of host tree selection and resistance can not be affected by variation of myrcene in the tree.


1Department of Animal Ecology, Lund University, SE-223 62 Lund, Sweden
Present address:
2Chemical Ecology, Göteborg University, SE-405 30 Göteborg, Sweden

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