Supplemental 3.
I. Determination of the enantiomeric ratios of pheromone components of D. frontalis and D.
mesoamericanus.
A) Frontalin and endo-Brevicomin. Volatiles collected from individual females of either species
that had fed singly in a log of Pinus oocarpa for 1 d were analyzed by enantioselective gas
chromatography. The samples were those for which non-enantiomeric pheromone production
data had been published previously (Sullivan et al., 2012). These insects had been collected
within the same general area (<15 km distance) as the insects used in the experiments of the
present study. The methods for sample collection are described on page 820 of Sullivan et al.
(2012); the results of the non-enantiomeric, quantitative analysis of detected pheromone
components in these samples are presented in Table 2 under the heading “Solitary in Log 1 d”.
Nine samples from D. mesoamericanus and 10 from D. frontalis were analyzed in splitless mode
on a Hewlett-Packard GC-MS (Model 6890-5973) fitted with a Supelco (Bellefonte,
Pennsylvania, United States of America) gamma-Dex 225 column (30 m length x 0.25 mm i.d. x
0.25 µm film thickness) which provided excellent separation of the enantiomers of frontalin and
endo-brevicomin. The oven program was 40° for 1 min, then 5°/min to 220°, then held 7 min.
Enantiomers were identified by matching mass spectra and retention times to those of
enantiomeric standards provided by Contech (previously PheroTech; Victoria, British Columbia,
Canada). Enantiomeric ratios (as percentage of the more abundant enantiomer) were determined
from the ratio of peak integration areas of single ion chromatograms of diagnostic ions for each
compound (for frontalin m/z 100; for endo-brevicomin m/z 114). Ratios were not corrected with
a dilution curve of known quantities of the enantiomers of the target compounds, however
previous analyses with the same instrument indicated that the ratio of mass to integration area
was approximately constant within the range of concentrations analyzed. (-)-Frontalin was the
dominant enantiomer for both D. mesoamericanus and D. frontalis females [D. mesoamericanus,
95.0% (±0.5% S.D.); D. frontalis, 94.3% (±0.9% S.D.)] whereas D. mesoamericanus females
produced >99% (+)-endo-brevicomin. Dendroctonus frontalis males sampled within the
southeastern United States of America also produce apparently >99% (+)-endo-brevicomin
(Sullivan et al. 2007).
B) Ipsdienol. Pooled subsamples of the volatiles collections of either D. mesoamericanus gallery
entrances (this study) or frass collected from the same females (manuscript in preparation) were
concentrated an additional 10-fold. These two pooled samples were then analyzed on a HewlettPackard model G1800C GC-MS fitted with a Supelco beta-Dex 120 capillary column (30 m
length x 0.25 mm i.d. x 0.25 µm film thickness) which permitted good resolution of the
enantiomers of ipsdienol. The oven program was 50° for 1 min, then 4°/min to 220° and held 10
min. Enantiomeric ratios were calculated from the single ion chromatogram of m/z 85. The
pooled frass aeration samples of female D. mesoamericanus contained approximately 98% (+)ipsdienol. The pooled entrance aeration samples of female D. mesoamericanus possessed a 3:97
ratio of the 85 m/z peak areas at the retention times of (-) and (+) ipsdienol, respectively;
however an apparent coeluent at the retention time of (-) may have inflated the peak area of this
enantiomer slightly. Consistent with these results, additional analyses of four samples of female
D. mesoamericanus mining in logs (samples reported in Sullivan et al. 2012) indicated that these
insects produced >95% (+)-ipsdienol.
II. Implications of use of racemic alpha-pinene, frontalin, and ipsdienol in assayed synthetic
blends:
(1) alpha-Pinene: The enantiomeric ratio of the alpha-pinene produced by the P. oocarpa logs
of the current study was not determined, but pines produce both (+) and (-)-alpha-pinene in
varying ratios (Klimetzek and Francke 1980, Marques et al. 2012). The racemic mixture seemed
to be a reasonable “default”, and use of the racemate was likely inconsequential to the
experiments since the alpha-pinene component was presented uniformly in all treatments.
(2) Frontalin: Availability determined our use of the racemate. Research on the behavioral
effects of the enantiomers of frontalin in D. frontalis has indicated that there are no antagonistic
affects between enantiomers and the (-)-enantiomer appears to be more attractive than the (+)enantiomer (Payne et al. 1982). In walking olfactometer assays, responses to a racemic mixture
did not differ significantly from responses to the (-)-enantiomer. When frontalin has been
shown to be an attractant for Dendroctonus (Wood et al. 1976, Phillips et al. 1990, Lindgren
1992), the enantiomers have not been found to be antagonistic although the (-)-appears to be the
more active enantiomer.
(3) Ipsdienol: Availability determined our use of the racemate. It is well-known that
enantiomeric ratio of ipsdienol can be highly specific and enantiomers can be antagonistic in Ips
DeGeer (Seybold 1993). Few Dendroctonus spp. have been found to produce ipsdienol (Skillen
et al. 1997), however a study on the effect of ipsdienol enantiomers on D. ponderosae Hopkins
[which like D. mesoamericanus produce the (+)-enantiomer)] indicated inhibitory effects on
response to attractants with generally no difference in activity between the (+)-enantiomer and
the racemate (Hunt and Borden 1988). In trapping experiments in Chiapas, (+)-ipsdienol
inhibited attraction by D. frontalis males whereas the (-)-enantiomer did not alter beetle
responses (authors’ unpublished data).
Hunt DWA, Borden JH (1988). Response of mountain pine beetle, Dendroctonus ponderosae
Hopkins, and pine engraver, Ips pini (Say), to ipsdienol in southwestern British
Columbia. J Chem Ecol 14: 277-293.
Klimetzek D, Francke W (1980) Relationship between the enantiomeric composition of α-pinene
in host trees and the production of verbenols in Ips species. Experientia 36: 1343-1345.
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