Supplementary Material
Extraction of Pheromone and Gas Chromatography-Mass Spectrometry
The pheromone gland of a female H. virescens was excised and extracted in 10 l of n-heptane
for at least 3 h prior to analysis. Extracts were analyzed by gas chromatography-mass
spectrometry (GC-MS) using a Hewlett-Packard 5890-5972 system, fitted with a 30 m x 0.25
mm i.d. ZB-Wax column (Phenomenex, Torrance, CA, USA). Samples were injected in the
splitless mode using helium as carrier gas. The GC oven was programmed from 80–180 at
15oC.min-1 (after a 1 min. delay), and then to 200 at 3oC.min-1. For determination of pheromone
titers, an internal standard of (E)-11-tetradecanal was used and the amount of the main
pheromone component, Z11-16:Ald (Roelofs et al., 1974) determined. The internal standard
was omitted from MIDA samples. The MS was operated in the selected ion monitoring mode,
monitoring m/z 192 (for the internal standard), and 220, 222, and 224 for the MIs of Z1116:Ald. The identity of Z11-16:Ald was confirmed by comparison of its mass spectrum and
retention time with those of an authentic standard. For titer determination, means were
determined from the analyses of 6-11 individual females. The TTR points (i.e., M+1 and M+2)
used in the calculations (and in Fig. 1) were derived from means of 5-9 individual females.
MIDA
The theory, assumptions, and calculations behind MIDA, as well as examples of application,
can be found in many reports (e.g., Chinkes et al., 1996; Hellerstein and Neese, 1999; Merchak
et al., 2000; Wolfe and Chinkes, 2005). Briefly, a stable isotope-labeled precursor is infused, or
given as a bolus, and the incorporation of label quantified in two or more mass isotopomers,
typically singly and doubly labeled (the incorporation of more than two precursor units is
usually below the detection limit of instrumentation), of a polymeric product. The measured
isotopomers must be corrected for isotopic contributions to their intensities from the unlabeled
(and from the singly to doubly labeled) isotopomer, before the precursor enrichment can be
calculated from the ratio of doubly to singly labeled isotopomers. Because the acetate
precursor (derived from U-13C-glucose) had two 13Cs, these contributions were relatively small.
Once the precursor enrichment is calculated, it is converted to units of singly labeled Z11-16:Ald
(i.e., the probability that a Z11-16:Ald molecule will have a single label) by a binomial equation,
enabling calculation of the FSR.
The various parameters were calculated thus:
The tracer: tracee ratio (TTR) of singly [TTR(M+1)] and doubly [TTR(M+2)] labeled Z1116:Ald were calculated by Eqns 1 and 2:
TTR(M  1)  (M  1 M  0) post  (M  1 M  0) pre Eqn 1


TTR(M  2)  (M  2  M  0) post  (M  2  M  0) pre  dT1  TTR(M  1) Eqn 2
Singly labeled Z11-16:Ald (M+1) has one 13C-labeled acetate and was measured at m/z 222,
while doubly labeled Z11-16:Ald (M+2) has two 13C-labeled acetates and was measured at m/z
224. Unlabeled Z11-16:Ald (M+0) was measured at m/z 220. The post terms are the measured
values following feeding on labeled glucose and the pre terms are the corrections for the
presence of naturally occurring stable isotopes (i.e., background). The corrections were made
(and reported) for theoretically calculated values. Experimental (using females feeding on
unlabeled glucose) background values were also determined (FSRs for Scot and Phot were,
0.954, 0.226, respectively), and were similar to the theoretical rates. The term dT 1 is the
percentage of singly labeled tracer with two heavy atoms, other than that from labeling from
one precursor (acetate) molecule, and allows for the contribution of the M+1 (i.e., m/z 222)
peak to the M+2 (m/z 224) peak.
The precursor isotopic enrichment (p), in molar percent excess (proportion of labeled
precursor), was calculated from the ratio of doubly to singly labeled product by Eqn 3:
p  [2  TTR(M  2)]  {[TTR(M  1)  (n 1)]  2  TTR(M  2)  TTR(M  1)} Eqn 3
where n = the number of precursor molecules in the product (i.e., 8 for Z11-16:Ald). This was

converted to units of enrichment of singly labeled Z11-16:Ald (Ep) by the binomial Eqn 4:
Ep  np(1 p) n1 Eqn 4

allowing calculation of the fractional synthetic rate by Eqn 5:
FSR  (Et2  Et1) [(T2  T1)  Ep] Eqn 5

where Et = singly labeled Z11-16:Ald enrichment at time t. The rate of change of singly labeled
Z11-16:Ald enrichment was derived from linear regression over the appropriate range (i.e.,
when it was increasing linearly after feeding) before isotopic steady state was reached. ASR was
calculated by multiplying FSR by the pool of Z11-16:Ald.
Additional references
CHINKES, D.L., AARSLAND, A., ROSENBLATT, J., and WOLFE, R.R. 1996. Comparison of mass
isotopomer dilution methods used to compute VLDL production in vivo. Am. J. Physiol.
Endocrinol. Metab. 271:E373-383.
HELLERSTEIN, M.K. and NEESE, R.A. 1999. Mass isotopomer distribution analysis at eight years:
theoretical, analytic, and experimental considerations. Am. J. Physiol. Endocrinol.
Metab. 276:E1146-1170.
MERCHAK, A., PATTERSON, B.W., YARASHESKI, K.E., and HAMVAS, A. 2000. Use of stable
isotope labeling technique and mass isotopomer distribution analysis of [13C]palmitate
isolated from surfactant disaturated phospholipids to study surfactant in vivo kinetics in
a premature infant. J. Mass Spectrom. 35:734-738.
ROELOFS, W.L., HILL, A.S., CARDÉ, R.T., and BAKER, T.C. 1974. Two sex pheromone components
of the tobacco budworm moth, Heliothis virescens. Life Sci. 14:1555-1562.