Supplemental Information
Methods
I. Analytical Procedures.
Temporal gland secretion (TGS) samples including over 100 from six male Asian
elephants in musth were initially frozen in liquid nitrogen in glass vials and stored at -80
o
C. Samples were analyzed by two gas chromatographic/ mass spectrometric (GC/MS)
protocols, after adsorption of headspace volatiles on a solid phase microextraction
(SPME) fiber, carboxan/polydimethylsiloxane. This fiber was exposed by lowering it
through a predrilled telfon septum so that the tip was 1 cm above 200 μl of TGS in a 1 ml
vial, immersed 1/3 in a 38oC water bath. Headspace volatiles were allowed to adsorb for
60min before the SPME fiber was lowered into the injection port [250 oC] of the gas
chromatograph (GC). In separate sequential analyses the compounds desorbed from the
fiber were separated by gas chromatography on first a DB-1 polymethyl silicone-coated
capillary column (60m x 0.25mm ID, 1.0 μm film thickness; J&W Scientific) using an
Agilent 6890 GC coupled to an Agilent mass spectrometer (MS) 5973 mass selective
detector (MSD). The mass spectrum of frontalin was compared with its published
spectrum1 and the spectrum obtained on our system with our synthesized standards of
racemic frontalin 2-6. Total frontalin was quantitated both on the GC/MS system by total
ion current (TIC) and on a GC equipped with a flame ionization detector (FID). To
resolve and quantitate the two enantiomers of frontalin the compounds desorbed from the
SPME fiber were separated by gas chromatography using a modified cyclodextrin as the
stationary phase (-cyclodextrin trifluoroacetyl capillary column, Alltech Chiraldex GTA,
30m x 0.25mm ID, 0.125 μm film thickness). Agilent 6890N GCs were coupled to a
Waters Micromass GCT mass spectrometer for New Zealand analyses and to an Agilent
MS 5973 MSD for USA analyses. Standards of our synthesized enriched (+) [1R,5S]
and enriched (–) [1S,5R] enantiomers 2-6 exhibiting 21% and 30% enantiomeric
enrichment respectively and 1H NMR, 13C NMR and MS spectra consistent with the
literature 6, as well as the synthesized racemic mixture were used to detect the retention
time of frontalin and were calibrated against internal standards of authentic synthetic
compounds of measured concentration. TIC values as well as the area values of m/z 142
and m/z 100 were measured to ensure accurate quantitation of frontalin as co-eluting
peaks were detected occasionally. The USA chiral system allowed an optional split mode
so that quantitation and identification could be confirmed, employing FID
simultaneously with MS.
II. Bioassay Procedures.
Based on the discovery of the presence and natural release of the two enantiomers of
frontalin and using the analyses of many banked TGS samples whose enantiomeric ratios
of frontalin had been established, we re-examined our data from previous assays of two
categories of males and three categories of females7, and performed new sets of assays
with three categories of mid-musth TGS (young teenage males in short “moda” musth,
older teenage males and males older than 25 years). These additional representative
paired bioassays using aliquots of previously analyzed TGS samples bioassays were
conducted using methods described in detail previously 7, 8. Five categories of elephant
conspecifics were tested: older males (21-43y), young males (13-20y), pregnant females,
females in the follicular stage of oestrus and females in the luteal phase. Chemosensory
responses monitored included sniffs, checks, places and flehmens; avoidance behaviours
scored included sample circling, ears erect, backing up, audible forceful exhalation, foot
scuffling, running away and accompanying vocalizations of trumpeting and roaring. For
precise definitions and expanded list see previous publications7-12. Parametric and
nonparametric statistics used included T-tests (Figure 1d) and one way analysis of
variance (ANOVA) on ranks (Figure 1a, b, and c), followed by all pairwise multiple
comparison procedures (Dunn’s Method)13. In Figure 1a, the statistically significant
differences (SSD) were between all groups, P < 0.05, Difference of Ranks (DR) =
(20.89–61.57), Q = (2.53–7.35). For Figure 1b similar statistical procedures were
employed; SSD, P < 0.05, were between 13–20y and 21–30y groups, and between 13–
20y and 31–43y groups respectively, DR = 15.51 and 18.94, Q = 2.59 and 2.49. For
Figure 1c similar statistical procedure as in Fig. 1a and 1b revealed SSD, P < 0.05,
between 6 groups as indicated by paired sequential numbers. [1-1] DR = 36.40, Q = 4.24;
[2-2] DR = 26.34, Q = 2.81; [3-3] DR = 43.52, Q = 3.96; [4-4] DR = 25.76, Q = 2.73; [55] DR = 29.32, Q = 3.16; [6-6] DR = 28.66, Q = 3.09. T-tests were employed for data in
Figure 1d. SSD, P = 0.001, were demonstrated between the following groups as indicated
by paired sequential numbers. The comparisons were between similar conspecific
groupings or within TGS-type assays. [1-1] Degrees of freedom (DF) =4, t = 4.66 [P =
0.01]; [2-2] DF = 4, t = 4.86; [3-3] DF = 4, t =8.61; [4-4] DF = 4, t = 8.9.
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