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EPICUTICULAR COMPOUNDS OF Drosophila subquinaria AND D. recens:
IDENTIFICATION, QUANTIFICATION, AND THEIR ROLE IN FEMALE MATE CHOICE
SHARON CURTIS1, JACQUELINE SZTEPANACZ2, ASHWIN RAMAKRISHNAN1,
BROOKE E. WHITE3, KELLY A. DYER3, HOWARD RUNDLE2*, AND PAUL MAYER1
Compound Identification
In general, chromatograph peaks were characterized initially by the presence of M+ ions in their
individual mass spectrum. Additional information on positioning of methyl groups or double
bonds was achieved from characteristic compound fragmentation patterns, coupled with
reference standards, mass spectral database comparisons, and comparison of ECL numbers with
previously published values in the literature, as described below. Peaks occurring at the same
ECL decimal places should correspond to molecules possessing double bonds in the same places,
and these are cited as fractional numbers (e.g., x.6) where differences in the integer of the ECL
numbers (i.e. x) relate to the overall carbon chain length (Bartelt et al. 1986).
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Methyl Alkanes
Compounds identified as methyl alkanes yielded characteristic mass spectral fragmentation
patterns and fractional ECL numbers (x.6) that correspond with previously published values in
other insects (Howard et al. 1978, 2003, Carlson and Yocom 1986, Everaerts et al. 2010). The 2methyl alkanes demonstrated a characteristic loss of the terminal methyl group (CH3), followed
by the loss of C3H7 (43 amu) (Fig S1; Howard et al. 1978). At higher carbon numbers (C33 and
C35), the 2-methyl alkane was replaced by an alkane at a lower ECL fractional number (x.4) and
at reduced concentration. Literature ECL values suggest this is an internally-methylated alkane
(Carlson and Yocom 1986, Brown et al. 1990). However, these longer-chain compounds did not
produce sufficient mass spectral data for assigning the position of the methyl group (likely due to
their relatively low abundances), although they did produce an alkane molecular ion peak. These
compounds did not co-elute with the linear alkane standard, eliminating the possibility of a linear
alkane. Cross-referencing of the fractional ECL number with literature values (Carlson and
Yocom 1986, Brown et al. 1990), the NIST Chemistry Web Book (webbook.nist.gov/chemistry)
and Pherobase (www.pherobase.com) suggests these compounds to be 5, 7 or 9 methyl alkane.
Without mass spectral evidence to determine the exact position of the methyl group, we classify
these as internally-methylated alkanes to distinguish them from the terminally methylated 2methyl alkanes identified above.
2
Fig. S1 Observed mass spectra for 2-methyl octacosane (FID peak 2, ECL 28.62). The insert
shows the chemical structure and NIST 11 MS database mass spectra for 2-methyl octacosane.
FID peak 5 (30.66) yielded a similar spectra, identifying it as 2-methyl triacontane
Alkenes
Alkenes were identified by the presence of M+ ions in their individual mass spectrum. Assigning
the position of a double bond in an alkene from mass spectral information alone is not possible
due to ion rearrangement. When available, fractional ECL numbers were cross-referenced with
published values (Howard et al. 2003) to predict double bond positions. Derivatization of the
double bonds in the alkenes (and alkadienes) was attempted using dimethyl disulphide (DMDS)
according to the procedures described in Carlson et al. (1989). The characteristic fragment ions
associated with the addition of DMDS were not present in the spectra, nor were the cyclic adduct
product ions normally generated when double bonds are separated by fewer than four methylene
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units. Derivatization may have failed due to the lower reactivity of these long chain length
compounds coupled with very low abundances (Carlson et al. 1989). A cis geometry has been
assumed for all alkenes given the results of previous investigations (Howard et al. 1978, 2003,
Everaerts et al. 2010).
Alkadienes
Alkadienes were identified by their M+ ions. Positions of the two double bonds have been
provisionally assigned based on their mass spectra ion fragment patterns. In particular, electron
impact fragmentation of alkadienes occurs at the double bonds and at the α- and β-carbons to the
double bonds, leaving stable ions. This disrupts the sequential loss of CH2 fragments at this
position. The resulting fragment ion is then able to fragment from both ends, creating plateaus of
sequential CH2 loss due to the slight increase in stability of these ions. When the next double
bond is encountered during the ionization process, the same progression occurs: a disruption in
sequential loss of CH2 producing a mass anomaly, followed by another plateau of stable ions
created by the sequential loss of CH2 from the terminal end not containing the double bond.
Figs. S2 and S3 show the disruption to the sequential loss of CH2 fragments from the
hydrocarbon chain upon ionization in the mass spectra for the provisionally identified 5-11
tritriacontadiene and 5-13 tritriacontadiene respectively. The inserted diagrams highlight
important fragment ions that would be created as the respective compound ionizes (shown in a
trans geometry for clarity). This mass disruption pattern, followed by a plateau of stable ions
until the next double bond is encountered, was observed in all of the alkadienes. The mass
spectra of these two compounds are very similar with the exception of the lower molecular
weight disruption and plateau of stable ions, which appears shifted to higher molecular weights
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in 5-11 tritricontadiene than in 5-13 tritriacontadiene, suggesting the second double bond in 5-11
tritriacontadiene is two carbons closer to the terminal end of the molecule.
Fig. S2 Mass spectral evidence for the assignment of 5-11 tritriacontadiene (FID peak 13, ECL
32.73). Inserts depict fragmentation ions from cleavage across the double bonds. FID peaks 6
(ECL 30.74) and 21 (ECL 34.74) yielded a similar mass spectral fragmentation pattern and
fractional ECL number (ECL x.74), suggesting they belong to a series of 5-11 alkadienes
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Fig. S3 Mass spectral evidence for the assignment of 5-13 tritriacontadiene (FID peak 12, ECL
32.66). Inserts depict fragmentation ions from cleavage across the double bonds. FID peak 20
(ECL 34.66) yielded a similar mass spectral fragmentation pattern and fractional ECL number
(x.66), suggesting it is also a 5-13 alkadiene
Published literature values for alkadienes in other insect species have fractional ECL numbers
lower than those analyzed here (Howard et al. 1978, 2003, Everaerts et al. 2010). However, a
comparison of boiling points for a range of C7 hydrocarbons illustrates how the positioning of
double bonds (and methyl branches) affects the boiling points of the molecules, which will in
turn alter their retention indices and thus their fractional ECL values (Table S1). Of particular
note is the fact that the two alkadienes (1, 2 vs. 1,5) have a boiling point difference of
approximately 10 °C, indicating the possibility for considerable differences in fractional ECL
numbers for different alkadienes depending on the placement of the double bonds.
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Table S1 Differences in boiling points of a range of C7 hydrocarbons (Owczarek and Blazej
2003)
Molecular formula
C7H16
C7H16
C7H16
C7H14
C7H14
C7H14
C7H14
C7H14
C7H14
C7H12
C7H12
C7H12
Molecule
heptane
2-methyl hexane
3-methyl hexane
1-heptene
trans-2-heptene
cis-2-heptene
trans-3-heptene
cis-3-heptene
2-methyl-1-hexene
1,2 heptadiene
1,5 heptadiene
3-heptyne
Boiling point (K)
371.57
363.15
364.99
366.80
371.06
371.56
368.81
368.90
364.65
376.90
366.85
380.35
Oxygen Containing Organic Compounds
11-cis-Vaccenyl acetate (FID peak 1, ECL 21.9) has been commonly observed in other insects
(see main text) and was identified in the current samples from its characteristic mass spectrum.
FID peaks 15, 16, 16a, 21-23 all have very similar mass spectra, suggesting they belong to a
single class of compounds. The GC:MS Wiley 275 data base suggested these were triacylglycerides, in particular glycerol trihexanoate. GC:MS analysis of standard glycerol
trihexanoate (Sigma Aldrich) produced a mass spectra similar to those observed in males from
both species (Fig. S4).
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Fig. S4 Mass spectrum from a male D. recens (above) and a standard C6 tri-acylglyceride,
glycerol trihexanaote (386 amu; below)
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In the mass spectrum of the standard glycerol trihexanaote, the M+ ion is not the most abundant
in the fragmentation pattern. Comparison of the ECL numbers for glycerol trihexanoate (ECL
24.38) and the sample peaks (ECL > 33) demonstrates that the tri-acylglycerides present in the
males contain longer fatty acid chains than the standard. This is supported by the analysis of the
derivatized free fatty acids (see the main text). While it is tempting to assign individual lengths
for the fatty acid chains based on the loss of an alkyl chain to produce the first noticeable peaks
within the mass spectrum, different fatty acid chain lengths and fragmentation patterns could
produce this spectra. Tri-acyl, di-acyl and mono-acyl glycerides have all been previously
identified in various insects (Lockey 1988; Ogg et al. 1991, 1993, Yew et al. 2011), and further
characterization of these polar compounds would therefore require an alternative technique such
as electrospray ionization mass spectrometry.
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Sexual selection analyses
Table S2. Eigenvectors of the loadings of 16 logcontrast CHCs on their principal components (PC), along with the percent variance
accounted for by each (% var)
Logcontrast
CHC (FID #)
% var
3
4
5
6
7
9
10
11
12
13
14
17
18
19
20
21
PC1
PC2
PC3
PC4
PC5
PC6
PC7
PC8
PC9
PC10
PC11
PC12
PC13
PC14
PC15
PC16
44.76
0.114
0.188
0.242
0.196
0.231
0.211
0.166
0.326
0.323
0.294
0.310
0.180
0.150
0.310
0.314
0.295
17.73
-0.294
-0.330
-0.127
-0.289
-0.288
0.311
0.322
0.120
-0.039
-0.067
-0.167
0.401
0.394
0.216
-0.102
0.027
7.6
0.578
0.385
0.020
0.137
0.104
0.151
0.334
-0.082
-0.172
-0.404
-0.224
0.124
0.248
-0.042
-0.153
-0.033
6.88
-0.121
-0.160
0.544
0.189
-0.202
-0.352
0.143
0.053
-0.097
-0.125
0.170
-0.306
0.405
-0.088
-0.251
0.240
5.43
0.195
-0.168
-0.135
0.470
-0.379
0.010
0.288
0.305
0.127
0.259
-0.084
-0.159
-0.227
0.128
-0.219
-0.379
3.81
-0.097
0.200
-0.043
0.495
-0.436
-0.089
-0.432
-0.144
-0.229
-0.058
-0.049
0.388
0.089
0.181
0.192
0.109
3.34
-0.509
0.365
-0.301
0.235
-0.035
0.115
0.455
-0.110
-0.045
-0.107
0.312
-0.068
-0.106
-0.307
0.025
0.087
2.43
0.275
-0.137
-0.519
-0.064
0.062
-0.467
0.149
0.000
-0.220
0.235
0.177
0.118
0.019
0.095
-0.153
0.454
2.06
0.224
-0.387
-0.005
0.195
-0.078
0.624
-0.119
-0.303
-0.096
0.072
0.145
-0.162
-0.089
-0.198
-0.086
0.379
1.39
-0.081
0.278
-0.111
-0.044
0.154
0.246
-0.317
0.216
-0.370
0.337
0.223
-0.124
0.346
0.037
-0.421
-0.239
1.19
0.024
-0.094
-0.383
0.182
0.082
-0.062
-0.243
-0.124
0.659
-0.203
0.100
-0.062
0.452
-0.088
-0.126
-0.093
1.13
-0.189
-0.397
0.035
0.424
0.617
-0.069
0.114
-0.191
-0.252
-0.051
-0.028
0.158
0.038
0.182
0.031
-0.253
0.78
0.149
-0.102
0.221
-0.036
-0.039
-0.093
0.001
0.005
0.077
0.131
0.341
0.623
-0.074
-0.545
-0.162
-0.228
0.64
0.220
-0.133
-0.111
-0.112
-0.184
-0.032
0.114
-0.113
-0.234
0.080
0.289
-0.214
0.340
-0.099
0.639
-0.353
0.46
-0.081
0.085
0.018
0.117
0.078
-0.047
0.088
-0.121
0.055
0.558
-0.601
-0.011
0.267
-0.406
0.130
0.115
0.38
0.015
0.180
0.165
-0.138
-0.121
-0.039
0.173
-0.725
0.169
0.309
0.129
0.016
-0.008
0.378
-0.208
-0.148
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