Zingerone in the flower of passiflora maliformis attracts an australian fruit fly, bactrocera jarvisi (Tryon)

Article abstract of DOI:10.3390/molecules25122877

Passiflora maliformis is an introduced plant in Australia but its flowers are known to attract the native Jarvis’s fruit fly, Bactrocera jarvisi (Tryon). The present study identifies and quantifies likely attractant(s) of male B. jarvisi in P. maliformis

Full text of DOI:10.3390/molecules25122877

Article
Zingerone in the Flower of Passiflora Maliformis
Attracts an Australian Fruit Fly, Bactrocera Jarvisi
(Tryon)
1
,
2
2
3
Soo Jean Park *, Stefano G. De Faveri , Jodie Cheesman , Benjamin L. Hanssen ,
1
3
1
Donald N. S. Cameron , Ian M. Jamie and Phillip W. Taylor
1
Applied BioSciences, Faculty of Science and Engineering, Macquarie University,
Sydney, NSW 2109, Australia; donald.cameron@mq.edu.au (D.N.S.C.); phil.taylor@mq.edu.au (P.W.T.)
Horticulture and Forestry Science, Queensland Department of Agriculture and Fisheries,
Mareeba, QLD 4880, Australia; stefano.defaveri@daf.qld.gov.au (S.G.D.F.); jodie.cheesman@daf.qld.gov.au
2
(J.C.)
3
Department of Molecular Sciences, Faculty of Science and Engineering, Macquarie University,
Sydney, NSW 2109, Australia; benjamin.hanssen@hdr.mq.edu.au (B.L.H.); ian.jamie@mq.edu.au (I.M.J.)
Correspondence: soojean.park@mq.edu.au; Tel.: +61-413-616-107
*
Academic Editor: Graham T. Eyres and Derek J. McPhee
Received: 5 May 2020; Accepted: 19 June 2020; Published: 22 June 2020
Abstract: Passiflora maliformis is an introduced plant in Australia but its flowers are known to attract
the native Jarvis’s fruit fly, Bactrocera jarvisi (Tryon). The present study identifies and quantifies
likely attractant(s) of male B. jarvisi in P. maliformis flowers. The chemical compositions of the inner
and outer coronal filaments, anther, stigma, ovary, sepal, and petal of P. maliformis were separately
extracted with ethanol and analyzed using gas chromatography-mass spectrometry (GC-MS).
Polyisoprenoid lipid precursors, fatty acids and their derivatives, and phenylpropanoids were
detected in P. maliformis flowers. Phenylpropanoids included raspberry ketone, cuelure, zingerone,
and zingerol, although compositions varied markedly amongst the flower parts. P. maliformis
flowers were open for less than one day, and the amounts of some of the compounds decreased
throughout the day. The attraction of male B. jarvisi to P. maliformis flowers is most readily explained
by the presence of zingerone in these flowers.
Keywords: passion fruit flower; Jarvis’s fruit fly; phenylpropanoids; raspberry ketone; cuelure; GC-
MS.
1
. Introduction
Mature males of many dacine fruit flies (Tephritidae) are attracted to the flowers of certain plants
and this attraction is often related to the presence of either methyl eugenol (4-allyl-1,2-
dimethoxybenzene) or raspberry ketone (4-(4-hydroxyphenyl)-2-butanone) in these flowers [1–6].
For example, the flowers of most Bulbophyllum orchids do not produce nectar, but instead produce
volatile compounds to attract pollinators [7], including Bactrocera fruit flies [7–9]. A broad range of
Bactrocera species is attracted to methyl eugenol and cuelure (4-(4-acetoxyphenyl)-2-butanone), a
more volatile analog of raspberry ketone [1,10,11]. These are the standard lures used in surveillance
and monitoring traps and for male annihilation techniques. Zingerone (4-(4-hydroxy-3-
methoxyphenyl)-2-butanone) contains common functional groups from both methyl eugenol and
cuelure, and the zingerone-containing flowers of Bulbophyllum patens attract a wide range of methyl
Molecules 2020, 25, 2877; doi:10.3390/molecules25122877
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2877
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eugenol and cuelure responsive flies [12]. Male flies that feed on methyl eugenol, raspberry ketone,
or cuelure are known to gain substantial increases in sexual performance [13–17].
Bactrocera jarvisi (Tryon) is a moderate pest fruit fly in Northern Australia, having a host range
of 83 species, of which 15 are cultivated [18]. The distribution of B. jarvisi is largely consistent with its
native host, Planchonia careya (Cocky apple) [19], extending down the east coast of Australia, to as far
south as Sydney, and across northern Australia into the Northern Territory and to Broome in Western
Australia [18,20]. While they exhibit little response to cuelure, B. jarvisi males are strongly attracted
to zingerone [21,22]. Bactrocera jarvisi males are attracted to the flowers of an Australian native orchid,
Bu. baileyi, which, like Bu. patens, contains zingerone [2,7,12]. The attraction of B. jarvisi to flowers of
the native tar tree, Semecarpus australiensis, and two exotic (American) passion fruits, Passiflora
maliformis and P. ligularis, has been reported [22,23], but compound(s) responsible for this attraction
have not been identified.
Passiflora maliformis is native to the Caribbean, Central America, and northern South America
[
24]. Although P. maliformis is not cultivated commercially, it does produce edible fruits [25–27]. The
seeds of P. maliformis contain essential fatty acids [25], and the fruit juice has antioxidant activity [26].
Chemical profiles of whole fruit extracts of P. maliformis have been studied, reporting six major and
3
2 minor components [28]. Headspace profiles of P. maliformis flowers are known to contain indole
as the major compound, bisabolene, geranyl acetone, 6-methyl-5-hepten-2-one, and (E)-β-ocimene
22]. However, the reported headspace profiles do not include phenylpropanoid compound(s) that
[
are known to attract B. jarvisi [22].
The flowers of P. maliformis open for only one day before senescence, and the authors have
observed that more B. jarvisi males are attracted to the coronal filaments of the flower in the morning
than later in the day. Male Bactrocera is commonly more responsive to lures in the early morning than
other times of the day [29–31], and so this observation may simply reflect diurnal patterns of B. jarvisi
lure response. However, it may also be that P. maliformis flowers release more attractant(s) in the
morning.
To ascertain the chemical basis for attraction of B. jarvisi to flowers of P. maliformis, a species with
which they do not share an evolutionary history, the present study (1) identifies compound(s) in P.
maliformis flowers that might attract male B. jarvisi, and (2) quantifies prospective attractant(s) in the
flowers through the day.
2
. Results
2
.1. Chemical Profiles of P. Maliformis Flowers
The identified compounds and their location in P. maliformis flowers are shown in Table 1. The
flowers contained terpenoids, C16 and C18 fatty acids and their derivatives, phenylpropanoids,
farnesol, farnesyl acetate, and squalene. Fatty acids and their derivatives and farnesol derivatives
were most abundant in the coronal filament extracts. Fatty acids and their derivatives are most
abundant in the reproductive organs, while terpenoids are most abundant in the petal and sepal.
Phenylpropanoids included raspberry ketone, cuelure, zingerone, and zingerol. These are present
but minor in the coronal filaments. Traces of raspberry ketone, cuelure, and zingerone were detected
in the petal and sepal. The compounds, 3-ethyl-2,3-dihydro-4H-pyran-4-one and 1-(4-methyl-1,3-
dioxolan-2-yl)pentan-3-one were tentatively identified (See Figure S1 and S2 in Supplementary
Materials).
2
.2. Diurnal Changes in Raspberry Ketone, Cuelure, Zingerone and Zingerol in P. Maliformis Coronal
Filaments
Four known fruit fly attractants, raspberry ketone, cuelure, zingerone, and zingerol were
detected. However, the compounds were not detected in all flower parts. The sepal and petal showed
traces of raspberry ketone, cuelure, and zingerone, but as the amount was below the limit of detection
of the generated standard curves, further quantification in these flower parts was not possible. Figure
1
illustrates changes in the amounts of the four compounds from 6 am to 5 pm AEST. Statistical

Molecules 2020, 25, 2877
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analysis showed that in the inner coronal filament, the amounts of raspberry ketone (F11,46 = 1.0, p >
.05), cuelure (F11,46 = 1.1, p > 0.05), and zingerol (F11,46 = 1.6, p > 0.05) did not change with flower
0
collection time, but the amount of zingerone changed through the day (F11,46 = 6.4, p < 0.0001). In the
outer coronal filaments, the amount of cuelure (F11,46 = 2.7, p < 0.01) and zingerone (F11,46 = 5.2, p <
0
.0001) changed through the day, but the amount of raspberry ketone (F11,46 = 1.1, p > 0.05) and zingerol
(
F11,46 = 1.5, p > 0.05) did not change. Further analysis of non-linear least-squares fitting showed that
the amounts of zingerone and cuelure decayed exponentially through the day (Table 2 and see
residual plots in Figure S3 in Supplementary Materials). Although the data of the raspberry ketone
in the inner coronal filament converged when fitted to the function, the fitting results showed that a
decay pattern was not strong (Table 2 and Figure S3 in Supplementary Materials). The data of
zingerol in both filaments, raspberry ketone in the outer coronal filament, and cuelure in the inner
coronal filament did not converge to fit the function.

Molecules 2020, 25, 2877
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Table 1. Chemical profiles of the seven assessed flower parts of P. maliformis. Relative abundances of chemicals were calculated by dividing the gas chromatography
GC) peak area of a compound by the sum of total peak areas of all compounds; The relative abundances were obtained from the 7 am samples; * Tentatively
(
identified compounds, further information is in the Supplementary Material; RI: Kovats retention index; Lit. RI: Literature retention index; ref: references for
retention index with most similar GC conditions.; MM: molecular mass; ICF: inner coronal filament; OCF: outer coronal filament; SE: Standard error.
Lit. RI
ref]
ICF/%
(SE)
OCF/% Anther/ Stigma/ Ovary/ Petal/%
Sepal/
% (SE)
25.39
(9.21)
1.78
(1.00) (
0.50
(0.20)
3.22
(1.74)
0.10
(0.04)
0.18
(0.06)
No
1
Identity
RI
980
MM
136.2
126.1
154.2
172.2
158.2
117.1
Diagnostic Ions
136, 121, 93, 79, 69, 41
126, 98, 81, 70, 53,
[
(SE)
% (SE)
% (SE)
% (SE)
(SE)
2
(
6.27
8.86)
1.21
β-Pinene
978 [32]
0.0
0.0
0.0
0.0
0.0
3
-Ethyl-2,3-dihydro-4H-
5.93
(1.96)
3.43
(1.08)
1.19
(0.74)
1.21
(0.77)
0.38
(0.06)
8.28
(3.43)
0.53
2
1042
0.0
0.0
pyran-4-one*
(0.69)
0.49
(0.11)
4.78
(2.00)
0.42
0
.52
3
Borneol
1175 1171 [33]
1227
154, 139, 123, 110, 95, 55, 41
0.0
0.0
(
0.12)
8.38
(3.33)
0.52
1
-(4-Methyl-1,3-dioxolan-
172, 143, 128, 99, 85, 82, 72,
57
8.91
(2.71)
5.77
(2.21)
0.12
2.57
(1.66)
1.03
4
2
-yl)pentan-3-one*
0
.20
0.04)
.16
6
Nonanoic acid
1278 1275 [34]
1295 1294 [35]
129, 115, 73, 60, 57, 41
(
(
(0.05)
0.13
(0.04)
(0.82)
0.09
(0.05)
(0.14)
(0.14))
0.09
(0.06)
(0.07)
0.19
(0.05)
0
7
Indole
117, 90
0.0
0.01)
4
-(2-Hydroxyisopropyl) -1-
methylcyclohexanol
2
.02
1.22
(0.63)
1.04
(0.71)
8
9
1315
172.3
139, 96, 81, 59, 43
0.0
0.0
0.0
0.0
(
1.04)
(
Terpin)
4
-(4-Hydroxyphenyl)-2-
butanone (Raspberry
ketone)
-(4-Acetoxyphenyl)-2-
butanone (Cuelure)
0
.22
0.11
(0.09)
0.05
(0.04)
0.06
(0.03)
1560 1556 [36]
164.2
206.2
194.2
164, 107
206, 164, 107
194, 137
0.0
0.0
0.0
0.0
0.0
0.0
0.0
(
0.04)
4
0.16
1.11
(0.30)
0.03
(0.02)
0.04
(0.02)
1
1
0
1
1652
1663
0.0
(0.06)
.10
0.24)
4
-(4-Hydroxy-3-
methoxyphenyl)-2-
butanone (Zingerone)
-(4-Hydroxy-3-
methoxyphenyl)-2-butanol 1688
Zingerol)
1
0.12
(0.07)
0.06
(0.02)
0.06
(0.02)
0.0
(
(
4
0
.22
1
1
1
2
3
4
196.2
242.4
222.4
196, 137
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.04)
(
2
42, 201, 183, 115, 102, 73,
1, 43
1.88
(1.00)
Propyl laurate
2E,6E)-3,7,11-
1690 1685 [37]
0.0
6
(
2
22, 136, 121, 107, 93, 81,
9, 41
5.87
(3.22)
3.92
(1.45)
Trimethyldodeca-2,6,10-
1732 1728 [38]
0.0
6
trien-1-ol (Farnesol)

Molecules 2020, 25, 2877
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3
,7,11-Trimethyl-2,6,10-
2
2
64, 204, 161, 136, 121, 107,
3, 81, 69, 43
7.78
(2.34)
8.31
(2.45)
0.42
(0.18)
1.76
(1.10)
1.02
(0.47)
0.58
(0.20)
0.52
(0.22)
1
5
dodecatrien-1-ol acetate
1849 1846 [39]
264.4
9
(Farnesyl acetate)
56, 312, 185, 171, 157, 129,
3, 57
8.76
(1.07)
17.82
(5.78)
2.33
(0.43)
0.24
(0.09)
2.10
(0.64)
4.32
(1.76)
15.23
(4.63)
2.21
(1.46)
2.10
(1.05)
12.27
(4.84)
19.08
(6.66)
0.08
(0.03)
8.28
12.52
(5.02)
14.89
(5.98)
3.86
(2.02)
4.42
(2.56)
5.11
(2.88)
19.45
(7.21)
9.52
(3.32)
3.83
(1.23)
13.47
(5.62)
15.08
(6.13)
5.03
(2.38)
5.86
(2.18)
4.63
(2.23)
17.94
(6.12)
9.44
(3.33)
6.52
(2.28)
7.78
(3.12)
4.85
(1.96)
1.82
(0.98)
4.33
(1.94)
2.82
(1.21)
16.36
(5.05)
3.70
(1.83)
1.84
(0.92)
8.42
(3.88)
5.32
(2.24)
11.45
(4.44)
5.78
(2.21)
3.61
(1.22)
16.50
(5.55)
3.82
(2.00)
1.79
(1.04)
1.02
(0.41)
9.35
1
1
1
1
2
2
2
2
2
2
6
7
8
9
0
1
2
3
4
5
Palmitic acid
1978 1977 [40]
1997 1996 [41]
2121 2117 [41]
2157 2156 [42]
2162 2162 [34]
2171 2171 [43]
2174 2173 [44]
2188 2188 [45]
2198 2197 [46]
2834 2833 [41]
256.4
284.5
296.5
280.4
278.4
308.5
306.5
284.5
312.5
410.7
7
6
.87
1.17)
.23
Ethyl palmitate
Phytol
284, 241, 157, 101, 88, 70, 55
(
(
0
123, 111, 97, 81, 71m, 69, 57
0.06)
2.38
(0.62)
1.83
(0.76)
2
80, 150, 138, 124, 109, 95,
1, 67, 55
78, 222, 149, 135, 121, 108,
3, 95, 79, 67, 55
Linoleic acid
Linolenic acid
Ethyl linoleate
Ethyl linolenate
Stearic acid
Ethyl sterate
Squalene
8
(3.35)
2.22
2
9
(1.08)
18.41
(6.33)
23.77
(7.45)
1.12
(0.32)
4.03
(2.72)
4.49
(2.15)
9
.54
308, 262, 109, 95, 81, 67, 55
06, 264, 149, 135, 121, 108,
5, 79, 67, 55
284, 241, 185, 129, 73, 60, 43
12, 269, 213, 157, 101, 88,
70, 55
67, 341, 203, 175, 161, 136,
21, 85, 81, 69
(
2.24)
2.03
3
9
(1.34)
.84
1
(
0.78)
2.13
(0.83)
33.83
(10.01)
3
1.43
6.02
(3.44)
4.74
4.77
(1.75)
5.65
0.72
6
(0.93)
32.58
(10.21)
(0.32)
19.85
(7.02)
3
1
(2.83)
(2.71)
(4.44)

Molecules 2020, 25, 2877
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Figure 1. Diurnal changes of raspberry ketone, cuelure, zingerone, and zingerol in the coronal
filaments of P. maliformis. Error bars represent standard errors that reflect variation across individual
flowers.
Table 2. Parameters from the curve fitting results for raspberry ketone, zingerone and cuelure. Fitted
−
푘×푥
function: 푓(푦) = 퐴 × 푒
+ 휀; A = Initial value for x (collection time); SE: standard error; k =
slope; ε = correction factor; T1/2 = estimated half-life; RSS = residual sum-of-squares; RSE = residual
standard error.
T
Hour
1/2
,
RSS
(RSE)
34.62
(0.793)
31.51
(0.756)
3454
(7.925)
Compound
Flower Part
A (SE)
ε (SE)
k (SE)
Raspberry
ketone
Inner coronal
filament
Outer coronal
filament
Inner coronal
filament
Outer coronal
filament
3429000
(6557000)
0.060
(0.116)
0.768
(0.112)
4.807
(2.127)
2.247
2.456
(3.662)
1.667
(1.789)
0.412
(1.331)
0.719
0
.282
Cuelure
4200 (245700)
0.964
0.416
1.682
3
65 (259)
Zingerone
563.4
(3.200)
9
68 (1166)
(0.575)
(1.307)

Molecules 2020, 25, 2877
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3
. Discussion
The biosynthetic intermediates for polyisoprenoid lipids, such as farnesol, farnesyl acetate, and
squalene, found in the flowers of P. maliformis are not unexpected as these are essential components
involved in the mevalonate pathway in plants [47]. This pathway is also associated with the synthesis
of terpenoids. Terpenoids, such as borneol, β-pinene, and terpin found in the current study, are
structurally diverse and are the most common plant secondary metabolites [48]. Borneol and both α-
and β-pinene are commonly found as volatile plant compounds [49–51]. Terpin is found in a bamboo
species [52]. Release of volatile terpenoids, such as those reported in the present study, is implicated
in the protection of plants against abiotic stresses, such as drought, high temperatures, and oxidative
stresses [53,54], as well as biotic interactions, such as herbivores or pathogenic attacks [55]. The
present study detected indole as a minor compound, while Fay reported it as the major compound
along with several other compounds [22]. This inconsistency might have been due to the different
sampling methods; the present study utilized solvent extraction of flower parts, while Fay entrapped
headspace from cut vines with flowers. Given that biosynthesis and accumulation of volatiles are
highly compartmentalized and restricted to specific tissues [56–58], and emission profiles can vary
with flower conditions during the sampling [59,60], the inconsistency is not unprecedented. The
occurrence of saturated and unsaturated fatty acids and their derivatives, including a suite of C18 fatty
acid derivatives, is ubiquitous in plants [61]. These acids are building blocks for very long-chain fatty
acids that are precursors for the synthesis of sphingolipids, important molecules involved in signal
transduction and regulation of cell death [62].
The detected phenylpropanoids, including raspberry ketone, cuelure, zingerone, and zingerol
in the coronal filaments confirms the previous speculation that the flower of P. maliformis may contain
zingerone that attracts B. jarvisi males [21,22]. Some volatile phenylpropanoids are emitted as floral
attractants to pollinators or as defense compounds against microorganisms, insects, and mammalian
predators [63–65]. Likewise, the phenylpropanoids found in this exotic plant are floral attractants to
Bactrocera species in Australia, although their functions in the plant’s native environment are not
known.
The production and emission of volatile compounds in plants increase during the early stages
of organ development and then either remain relatively constant or decrease over the organs’ lifespan
[
64]. This trend was observed in the present study in that the amounts of zingerone in both coronal
filaments and cuelure in the outer coronal filament decayed exponentially, and that of the other
compounds remained unchanged or did not show a strong decay pattern. The release of floral
volatiles is also controlled by circadian clock [66,67], and environmental factors, such as light,
temperature, and humidity [68]. The overall amounts of zingerone detected in the corona were higher
in the inner filaments than in the outer filaments (Figure 1). Cuelure was known only as a synthetic
attractant [1] until recent studies reported the occurrence of cuelure in nature [69–71]. The present
study also demonstrates cuelure as a naturally occurring compound. If the flowers released sufficient
cuelure and raspberry ketone, then cuelure-responding species should be attracted. The amount of
cuelure and raspberry ketone in the flowers was barely detectable, which likely explains why the
flowers have not been reported to attract cuelure-responding species. The attraction of B. jarvisi to
zingerone is as strong as that of B. dorsalis to methyl eugenol [21]. The efficacy of cuelure to cuelure-
responding species is not as strong as that of methyl eugenol to methyl eugenol responding species
[
72,73]. In combination with the very low levels of cuelure and raspberry ketone in the flowers, the
weaker efficacy of cuelure and raspberry ketone may also explain why species that are attracted to
these compounds have not been reported on P. maliformis flowers.
The release of floral volatiles in plants often coincides with the foraging activities of potential
pollinators and is usually regulated by light [64]. Although there is variation in insect pollination
between Passiflora species [74], generally, the pollinators for Passiflora include bees, hummingbirds,
and bats [75–77]. The corona of Passiflora is known to act as a visual and olfactory stimulus to its
pollinators [78]. Substances in the corona of Passiflora, including phenylpropanoids, may play a role
in attracting pollinators. The diurnal patterns of zingerone and cuelure contents of P. maliformis
flowers coincide with the typical diurnal pattern of responsiveness of Bactrocera males, which are

Molecules 2020, 25, 2877
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usually more responsive to phenylpropanoid attractants in the early morning [29–31]. Further study
is needed to confirm whether peak responsiveness of B. jarvisi to phenylpropanoids corresponds with
the timing of the greatest amounts of phenylpropanoids in P. maliformis. However, such
correspondence cannot be explained in evolutionary terms, as the natural range of P. maliformis does
not coincide with that of any known Bactrocera species. The functional significance of zingerone and
other phenylpropanoids in P. maliformis flowers and its significance to pollinators in the natural range
of this plant is currently unclear.
In summary, the present study finds a cause of interaction between an exotic plant, P. maliformis
and an Australian native fruit fly, B. jarvisi to be most likely by plant-produced zingerone, a volatile
compound that is highly attractive to B. jarvisi and is also released by flowers of some native plants
to attract insect pollinators.
4
. Materials and Methods
4
.1. Chemicals
For positive identification of the compounds in P. maliformis flowers, β-pinene, borneol, indole,
terpin, raspberry ketone (4-(4-hydroxyphenyl)-2-butanone), cuelure (4-(4-acetoxyphenyl)-2-
butanone), zingerone (4-(4-hydroxy-3-methoxyphenyl)-2-butanone), farnesol, palmitic acid, ethyl
palmitate, phytol, linolenic acid, ethyl linoleate, ethyl linolenate, stearic acid, ethyl stearate, and
squalene were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Nonanoic acid
was purchased from Acros Organics (Thermo Fisher Scientific, Geel, Belgium). Propyl laurate was
purchased from AbovChem (AbovChem LLC, San Diego, CA, USA). Farnesyl acetate (a mixture of
isomers) was purchased from TCI (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan). Linoleic acid
was purchased from Alfa-Aesar (Thermo Fisher Scientific, Lancashire, UK). The chemicals were
analytical or reagent grade with 98% or higher purity and used without further purifications.
Zingerol (4-(4-hydroxy-3-methoxyphenyl)-2-butanol) was synthesized by the reduction of zingerone
with sodium borohydride in methanol (See Supplementary Materials for synthetic details).
4
.2. Flower Extraction
Flowers of P. maliformis (Voucher number: CNS 147961.1, Australian Tropical Herbarium) were
collected between January 2017 and October 2018 at the Mareeba Research Facility of the Queensland
Department of Agriculture and Fisheries (17.007007 °S, 145.429446 °E). The flowers were collected
hourly from 6 am to 5 pm AEST with at least three replicates at each hour. Following collection, the
inner and outer coronal filaments, anther, stigma, ovary, sepal, and petal were separated (see Figure
S4 in Supplementary Material for the organization of the flower organs), cut into fine pieces and
stored in absolute ethanol (1 mL) in 2 or 4 mL glass vials at 4 °C until further extraction. Scissors and
forceps were decontaminated with 100% ethanol in between dissecting and cutting the separated
flower parts. The ethanol extracts were partitioned between aqueous and organic layers by adding
water (1 mL) and ethyl acetate in hexane (10% (v/v), 2 mL). The organic layer was separated, and the
aqueous layer was further extracted with ethyl acetate in hexane (10% (v/v), 2 mL × 2) using a
separatory funnel. The organic layers were combined, washed with deionized water (5 mL), dried
over anhydrous sodium sulfate, and concentrated under reduced pressure. Where necessary, the
concentrate was diluted with dichloromethane to give a sample volume of 200 µL that was subjected
to gas chromatography-mass spectrometry (GC-MS) analysis.
4
.3. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis was performed on a Shimadzu GCMS QP2010 or Shimadzu GCMS TQ8030
spectrometer equipped with a split/splitless injector, fused silica capillary column (SH-Rtx-5MS, 30
m  0.25 mm I.D.  0.25 μm film thickness) with crossbond 5% diphenyl/95% dimethyl polysiloxane
as the stationary phase and integrated mass spectrometry (MS). Helium gas (BOC, North Ryde, NSW,
Australia) (99.999%) was used as a carrier gas with a constant flow of 1.0 mL/min. The injection was
1
μL of a sample at 270 °C. The initial column temperature was set at 60 °C and held for 4 min,

Molecules 2020, 25, 2877
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increased to 200 °C at a rate of 10 °C/min, then increased to 300 °C at a rate of 40 °C/min, and held for
min. The temperatures of the interface and ion source box were set at 250 and 230 °C, respectively.
7
The ionization method was electron impact at a voltage of 70 eV. The spectra were obtained over a
mass range of m/z 41–650. The data were processed by the Shimadzu GCMS Postrun software. For
identification, mass spectra were compared with the NIST library (NIST17-1, NIST17-2, NIST17s) to
identify related molecules. The fragmentation patterns and retention indices published in the
literature were used to suggest candidate molecules. The proposed molecules were purchased, and
GC-MS analyzed using the same instrumental method. The identity of a compound was confirmed
by comparing retention time and fragmentations of the authentic molecule. The used solvents,
including absolute ethanol, hexane, ethyl acetate, and dichloromethane (DCM) were routinely
subjected to GC-MS runs to effectively identify and eliminate impurities.
4
.4. Quantification of Known Bactrocera Attractants in P. Maliformis
The amounts of raspberry ketone, cuelure, zingerone, and zingerol in the collected flower parts
were estimated by reference to standard curves. Stock solutions of raspberry ketone, cuelure,
zingerone, and zingerol were prepared in volumetric flasks (5 mL), and a series of standard solutions
were prepared by serial dilution of the stock solutions. An aliquot of 1.0 μL internal standard solution
(
tridecane, 1.06 mg/mL) was added to the standards and samples to give a final concentration of 5.30
µg/mL. The standard and sample solutions were run through GC-MS, and the peak area ratios of the
analytes to the internal standard were plotted against ratios of known concentrations of the analytes
to the internal standard to obtain standard curves. The concentration of a sample was estimated using
the slope of the standard curve, and the amounts of these compounds were subsequently estimated
using the original volume of a sample for each flower part.
4
.5. Chromatographic Purification of Unknown Compounds
Following the analyses of the flower parts, all of the corona sample solutions were pooled, and
the solvent was evaporated under reduced pressure. The residue was separated by double flash
column chromatography with ethyl acetate/hexane (0–20% ethyl acetate in hexane (v/v), gradient) as
a solvent system to give 3-ethyl-2,3-dihydro-4H-pyran-4-one and 1-(4-methyl-1,3-dioxolan-2-
yl)pentan-3-one, where the chromatographic fractions were monitored by GC. The NMR spectra of
the compounds were obtained to support the identities of the compounds.
4
.6. Data Analysis
The data were not normally distributed, and hence were log transformed for statistical analysis.
However, the raw data were used to generate graphs. The change in the amounts of the compounds
present in the flower tissues through the day were analyzed by a linear model to see whether the
amount of a compound changes with flower collection time. In the analysis, collection time was the
independent variable, the detected mass of compound was the dependent variable. Some of the
amounts of the compounds were significantly different throughout the day. To compare the amount
of a compound between collection times, normally, a posthoc analysis can be followed. Instead, we
were interested in decay patterns, and hence further analyzed by a non-linear curve fitting method
with the Function (1).
푓(푦) = 퐴 × 푒−푘×푥 + 휀,
(1)
Where, A = Initial value for x, k = slope, and ε = correction factor. Data were analyzed using R (v3.6.1.,
R Core Team, 2019).
Supplementary Materials: The following are available online, Figure S1. Mass spectrum and diagnostic ions of
3
2
-ethyl-2,3-dihydro-4H-pyran-4-one; Figure S2. Mass spectrum and diagnostic ions of 1-(4-methyl-1,3-dioxolan-
-yl)pentan-3-one, Figure S3. Residual plots of raspberry ketone in the inner coronal filament, zingerone in both
filaments, and cuelure in the outer coronal filament from exponential decay fitting, Figure S4. Flower of P.
maliformis and B. jarvisi males visiting.

Molecules 2020, 25, 2877
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Author Contributions: Conceptualization, P.W.T., S.G.D.F. and S.J.P.; methodology, S.J.P., S.G.D.F. and J.C.;
formal analysis, S.J.P.; investigation, S.J.P., J.C., B.L.H. and D.N.S.C.; resources, I.M.J. and P.W.T.; writing—
original draft preparation, S.J.P.; writing—review and editing, P.W.T., S.G.D.F., I.M.J., B.L.H. and D.N.S.C.;
supervision, P.W.T.; funding acquisition, P.W.T. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported by funds from the Australian Research Council Industrial
Transformation Training Centre for Fruit Fly Biosecurity Innovation (Project IC150100026), including a Research
Fellowship for S.J.P. This research was co-supported by the Queensland Department of Agriculture and Fisheries
(QDAF).
Acknowledgments: The authors gratefully acknowledge Nazma Akter Tithi for helping the preparation of
samples.
Conflicts of Interest: The authors declare no conflict of interest.
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