Attraction and electrophysiological response to identified rectal gland volatiles in bactrocera frauenfeldi (schiner)

Article abstract of DOI:10.3390/molecules25061275

Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) is a polyphagous fruit fly pest species that is endemic to Papua New Guinea and has become established in several Pacific Islands and Australia. Despite its economic importance for many crops and the key role of chemical-mediated sexual communication in the reproductive biology of tephritid fruit flies, as well as the potential application of pheromones as attractants, there have been no studies investigating the identity or activity of rectal gland secretions or emission profiles of this species. The present study (1) identifies the chemical profile of volatile compounds produced in rectal glands and released by B. frauenfeldi, (2) investigates which of the volatile compounds elicit an electroantennographic or electropalpographic response, and (3) investigates the potential function of glandular emissions as mate-attracting sex pheromones. Rectal gland extracts and headspace collections from sexually mature males and females of B. frauenfeldi were analysed by gas chromatography-mass spectrometry. Male rectal glands contained (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane as a major component and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane as a moderate component. Minor components included palmitoleic acid, palmitic acid, and ethyl oleate. In contrast, female rectal glands contained (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ethyl laurate as major components, ethyl myristate and ethyl palmitoleate as moderate components, and 18 minor compounds including amides, esters, and spiroacetals. Although fewer compounds were detected from the headspace collections of both males and females than from the gland extractions, most of the abundant chemicals in the rectal gland extracts were also detected in the headspace collections. Gas chromatography coupled electroantennographic detection found responses to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane from the antennae of both male and female B. frauenfeldi. Responses to (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane were elicited from the antennae of females but not males. The two spiroacetals also elicited electropalpographic responses from both male and female B. frauenfeldi. Ethyl caprate and methyl laurate, found in female rectal glands, elicited responses in female antennae and palps, respectively. Y-maze bioassays showed that females were attracted to the volatiles from male rectal glands but males were not. Neither males nor females were attracted to the volatiles from female rectal glands. Our findings suggest (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane as components of a sex-attracting pheromone in B. frauenfeldi.

Full text of DOI:10.3390/molecules25061275

molecules
Article
Attraction and Electrophysiological Response to
Identified Rectal Gland Volatiles in Bactrocera
frauenfeldi (Schiner)
Saeedeh Noushini 1,2,
*
, Jeanneth Perez 2,3 , Soo Jean Park 2,3, Danielle Holgate 1,
Vivian Mendez Alvarez 2,3, Ian Jamie 1,2 , Joanne Jamie 1,2 and Phillip Taylor 2,3
1
Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia;
danielle.holgate@hdr.mq.edu.au (D.H.); ian.jamie@mq.edu.au (I.J.); joanne.jamie@mq.edu.au (J.J.)
Australian Research Council Industrial Transformation Training Centre for Fruit Fly Biosecurity Innovation,
2
Macquarie University, Sydney, NSW 2109, Australia; jeanneth.perez@mq.edu.au (J.P.);
soojean.park@mq.edu.au (S.J.P.); vivian.mendez@mq.edu.au (V.M.A.); Phil.Taylor@mq.edu.au (P.T.)
Applied BioSciences, Macquarie University, Sydney, NSW 2109, Australia
3
*
Correspondence: sally.noushini@mq.edu.au
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 February 2020; Accepted: 7 March 2020; Published: 11 March 2020
Abstract: Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) is a polyphagous fruit fly pest species
that is endemic to Papua New Guinea and has become established in several Pacific Islands and
Australia. Despite its economic importance for many crops and the key role of chemical-mediated
sexual communication in the reproductive biology of tephritid fruit flies, as well as the potential
application of pheromones as attractants, there have been no studies investigating the identity
or activity of rectal gland secretions or emission profiles of this species. The present study (1)
identifies the chemical profile of volatile compounds produced in rectal glands and released by
B. frauenfeldi, (2) investigates which of the volatile compounds elicit an electroantennographic or
electropalpographic response, and (3) investigates the potential function of glandular emissions
as mate-attracting sex pheromones. Rectal gland extracts and headspace collections from
sexually mature males and females of B. frauenfeldi were analysed by gas chromatography-mass
spectrometry. Male rectal glands contained (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane as
a major component and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane as a moderate component.
Minor components included palmitoleic acid, palmitic acid, and ethyl oleate. In contrast,
female rectal glands contained (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ethyl laurate
as major components, ethyl myristate and ethyl palmitoleate as moderate components, and
18 minor compounds including amides, esters, and spiroacetals. Although fewer compounds
were detected from the headspace collections of both males and females than from the gland
extractions, most of the abundant chemicals in the rectal gland extracts were also detected
in the headspace collections. Gas chromatography coupled electroantennographic detection
found responses to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane from the antennae of both
male and female B. frauenfeldi. Responses to (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane
were elicited from the antennae of females but not males. The two spiroacetals also elicited
electropalpographic responses from both male and female B. frauenfeldi. Ethyl caprate and
methyl laurate, found in female rectal glands, elicited responses in female antennae and palps,
respectively. Y-maze bioassays showed that females were attracted to the volatiles from male
rectal glands but males were not. Neither males nor females were attracted to the volatiles from
female rectal glands. Our findings suggest (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and
(E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane as components of a sex-attracting pheromone in
B. frauenfeldi.
Keywords: B. frauenfeldi; mango fruit fly; insect volatiles; GC-EAD; olfaction
Molecules 2020, 25, 1275; doi:10.3390/molecules25061275
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1275
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1. Introduction
Many tephritid fruit flies, belonging to the genus Bactrocera, are significant horticultural pests,
causing direct damage to fruit and impeding trade [ ]. The mango fruit fly, Bactrocera frauenfeldi
1–4
(Schiner) (Diptera: Tephritidae), is an economically important tephritid pest, with hosts including
guava (Psidium guajava), mango (Mangifera indica), beach almond (Terminalia catappa), and Alexandrian
laurel (Calophyllum inophyllum) [
established on several Pacific Islands, including the Solomon Islands, the Federated States of Micronesia,
the Republic of Kiribati, Marshall Islands, Palau, Nauru, West Papua in Indonesia [ 11], and in
Australia [ 12 15]. Surveillance, monitoring, and control of tephritid fruit flies involves the use of
5–7]. Endemic to Papua New Guinea [8], B. frauenfeldi has become
9
–
6, –
lures [16]. Food-based lures have been used as attractants in traps for detecting and monitoring
tephritid flies [17]. However they are not as powerful as male lures, such as cuelure, zingerone, and
methyl eugenol [18]. Bactrocera frauenfeldi adult males are highly attracted to cuelure and raspberry
ketone, and are weakly attracted to zingerone [15
,18]. Raspberry ketone and zingerone are naturally
occurring compounds found in many plants [19
,
20]. Raspberry ketone is also known as a fungal
metabolite [21]. While cuelure has not been found in nature, it hydrolyses to raspberry ketone [16].
Ingested raspberry ketone, zingerone, and cuelure accumulate in the rectal gland of some male s
of some fruit fly species in their original form, while zingerone and cuelure are also to some extent
transformed [22–24]. However, similar to other fruit flies, there is a lack of a specific attractant for
females of B. frauenfeldi.
Volatile compounds produced in the rectal glands of tephritid fruit flies and emitted during
calling and courtship have been described as the key elements for long and short-range attraction
of the opposite sex [25
same sex to mating aggregations [29
studied for potential applications as attractants, including B. tryoni (Froggatt) [31], Zeugodacus cucumis
(French) [32 35], B. dorsalis (Hendel) [36], Z. cucurbitae (Coquillet) [37], B. oleae (Rossi) [38], and B.
–
28]. The volatile emissions also play an important role in attracting the
,
30]. Rectal gland secretions of some fruit fly species have been
–
correcta (Bezzi) [39]. In most tephritid fruit flies, males are thought to produce sex pheromones to attract
females [40]. However, there are several notable exceptions. For example, 1,7-dioxaspiro[5,5]undecane
has been described as a female-produced pheromone of B. oleae [38], although later studies reported on
this compound in rectal glands of young males of B. oleae [41]. While 1,7-dioxaspiro[5.5]undecane has
been used extensively for the monitoring and mass trapping of B. oleae [42], the sex specific olfactory
cues of B. oleae are driven by synergistic actions of a number of compounds that are not yet fully
understood [4,43]. In Z. cucurbitae, females are attracted by male rectal gland secretions containing three
aliphatic amides, two pyrazines, and an aromatic acid [44], while males are attracted by headspace
constituents containing 2,8-dialkyl-1,7-dioxaspiro[5.5]undecanes and N-(3-methylbutyl)acetamide [45].
Similarly, males of B. dorsalis produce two phenols and an aliphatic cyclic alcohol in their rectal glands
that show pheromonal activity towards females [46,47], while females emit several spiroacetals that
attract males [45].
The chemical profiles of the B. frauenfeldi volatile compounds are unknown. Given the central role of
chemical-mediated sexual communication in the reproductive biology of tephritid fruit flies [ 48], and
4,
the potential application of volatiles as attractants, the present study analysed the rectal gland extracts
and headspace collections from both males and females of B. frauenfeldi using gas chromatography-mass
spectrometry (GC-MS). In order to identify electrophysiologically active components present in the
emissions of each sex, we used gas chromatography-electroantennogram detection (GC-EAD) and
gas chromatography-electropalpogram detection (GC-EPD) to test the responses of male and female
antennae and maxillary palps to the emissions of male and female rectal glands. The rectal gland
contents were tested for attraction of the opposite and same sex using y-maze olfactometers [49–51].

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2. Methods and Materials
2.1. Insects
Pupae of B. frauenfeldi (9 generations from wild) were obtained from the Queensland Department
of Agriculture and Fisheries (Cairns, Australia). Approximately 500 pupae were allowed to emerge in a
47.5
room (25
×
47.5
×
47.5 cm fine mesh cage (Megaview Bugdorm 4S4545, Taiwan) in a controlled environment
◦
±
0.5 C, 65
±
5% relative humidity) and with light:dusk:dark:dawn 11.5:0.5:11.5:0.5 h
photoperiod (these conditions were maintained for all rearing and experiments). The adult flies
were fed sugar and yeast hydrolysate (MP Biomedicals LLC) and were provided water through a
soaked sponge. The flies were reared through one generation at Macquarie University, Sydney, using
a standard carrot larval diet [52], following methods described by Pérez et al. [53]. The flies were
separated by sex within three days after emergence and transferred to 12.5 L clear plastic cages (180 flies
per cage). No mating was observed before separating the flies. The flies used for all the experiments
were 13–18 days old.
2.2. Gland Extraction
The flies were killed by chilling them on dry ice. The rectal glands were extracted under a
stereomicroscope by gently pressing the abdomen and pulling the gland out with fine forceps. The
glands were carefully placed in a 1.1 mL tear-drop vial in dry ice. Once 10 glands were collected, the
vials were removed from the dry ice and 100
µL of n-hexane (HPLC grade, Sigma-Aldrich, St. Louis,
MO, USA) was added. Vials containing n-hexane and glands were left to stand at room temperature
for 10 min, and then the extracts were transferred to a new vial, labelled, and stored at
analysed [54]. Ten replicates of 10 glands were collected for each sex.
−
20 ◦C until
2.3. Headspace Collections
Based on our preliminary observations, B. frauenfeldi appeared to mate at any time during the
day, with the mating peak at noon. Ten males or females were separately placed into a cylindrical
glass chamber (150 mm long and 40 mm ID) 30 min before the mating peak. A charcoal-filtered air
stream at a flow rate of 0.5 L/min (air pulling system) was drawn over the flies for 1 h. The released
volatiles were adsorbed onto traps of 50 mg of Tenax-GR Mesh 60/80 adsorbent (Scientific Instrument
Services, Inc, Palmer, MA, USA) packed into 6
The volatiles were eluted with 1 mL of n-hexane. The samples were stored at
×
50 mm glass tubes and fitted with glass wool plugs.
20 ◦C until analysed.
−
Seven replicates were collected for each sex. To distinguish any possible contaminants, an air control
sample comprising an empty glass chamber was run and analysed along with each volatile collection.
Before each headspace collection, the glass chambers were washed with 5% Extran aqueous
solution, rinsed with hot tap water, and heated at 200 ◦C for 18 h. The tenax traps were thermally
conditioned at 200 ◦C for three hours under a 75 mL/min nitrogen stream. The activated charcoal filters
were conditioned by heating them at 200 ◦C for 18 h prior to each headspace collection [55].
2.4. GC-MS Analysis
Mass spectra were recorded on a Shimadzu GCMS-TQ8040 instrument (Kyoto, Japan), using a
capillary column with 5% diphenyl/95% dimethyl polysiloxane as the stationary phase (SH-Rtx-5MS,
30 m
×
0.25 mm ID
×
0.25 µm film thickness, Shimadzu, Japan) and helium (99.999%) (ultra-high
purity, BOC, Sydney, Australia) as a carrier gas with a constant flow of 1 mL/min. A 1
µL sample
◦
was injected in the splitless mode. The injector temperature was set at 270 C. The temperature
program was 40 ◦C (1 min) to◦250 ◦C (3 min) at a rate of 10 ◦C/min. The interphase and ion source
◦
temperatures were set at 290 C and 200 C, respectively. Mass spectra were recorded in electron
impact mode (70 eV), scanning from 40 to 500 m/z. A peak was considered of interest if it was
not present in the air control samples. Compounds including esters, amides, and spiroacetals were
identified through comparison with retention times and fragmentation patterns of authentic samples,

Molecules 2020, 25, 1275
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with the exception of compound
4
which was tentatively identified based on literature mass spectral
fragmentation patterns [56
,
57]. Ethyl caprate (
), methyl myristate (10), myristic acid (11), ethyl myristoleate (12), ethyl myristate
13), methyl palmitoleate (14), methyl palmitate (15), palmitoleic acid (16), palmitic acid (17), ethyl
5), methyl laurate (6), ethyl laurate (7), ethyl tridecanaote
(8), propyl laurate (9
(
palmitate (19), methyl elaidate (20), and ethyl oleate (21) were purchased from Sigma-Aldrich (Castle
Hill, Australia), Alfa-Aesar (United Kingdom), Nu-Chek-Prep, and INC (Minneapolis, USA). Ethyl
palmitoleate (18), ethyl elaidate (22), N-(2-methylbutyl)acetamide (
1), N-(3-methylbutyl)acetamide (
2),
and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5,5]undecane (3) were synthesised (see Supplementary Materials
for synthesis details).
2.5. Electrophysiological Assays
Gas
chromatography-electroantennographic
detection
(GC-EAD)
and
gas
chromatography-electropalpogram detection (GC-EPD) were carried out to identify the antennal- or
palpal-active components from male and female rectal gland extracts. The system consisted of a gas
chromatography flame ionization detector (Agilent 7890B, CA, USA) coupled to an electroantennogram
(Syntech, Hilversum, The Netherlands). The GC was equipped with a polar capillary column with
(35%-phenyl)-methylpolysiloxane as the stationary phase (Agilent HP-5, 30 m
×
0.32 mm ID
×
0.25 µm
film thickness). The carrier gas was hydrogen (99.999% pure) supplied by a generator (MGG-2500-220
Parker Balston, NY, USA) with a constant flow of 2.5 mL/min. The initial temperature was set at 50 ◦C
(1 min) then increased to 250 ◦C (3 min) at a rate of 10 ◦C/min. The injector and detector temperatures
were set at 270 ◦C and 290 ◦C, respectively. The effluent of the column was mixed with 30 mL/min
make-up nitrogen gas and split in a ratio of 1 (FID) to 1.5 (EAD) through a heated transfer line (Syntech,
TC-02, Syntech, Hilversum, The Netherlands) and kept at 200 ◦C.
A female or male B. frauenfeldi head was carefully severed and a borosilicate glass capillary
electrode filled with electrically conductive gel (Spectra 360) was attached onto the back of the head.
The tip of the antenna or maxillary palp was inserted into the tip of the recording glass capillary
electrode filled with phosphate-buffered saline (PBS). The mounted heads were under charcoal
filtered and humidified air flow (400 mL/min) controlled by a flow controller (Syntech Stimulus
Controller CS-55, Syntech, Hilversum, The Netherlands) and were subjected to each stimulus. The
electrophysiological responses were captured and processed by a data acquisition controller (IDAC-4,
Syntech, Hilversum, The Netherlands). Before the injection of the sample into the airstream, the
antenna/palp was stimulated with 1-hexanol to check its sensitivity. EAD/EPD signals were analysed
using GC-EAD 2014 software version 1.2.5. Nine successful GC-EAD/EPD recordings were obtained for
each sex. The electrophysiological responses of male and female antennae and palps to the conspecific
opposite and same sex rectal gland extracts were recorded. A response was considered genuine if it
was present in at least six out of the nine replicates collected. The identity of the compounds eliciting
an electrophysiological response was confirmed by comparing the retention times with that of the
GC-MS chromatograms using the same column and method as for the GC(FID)-EAD experiments.
2.6. Y-maze Bioassays
The response of sexually mature (13–18 days old) B. frauenfeldi males and females toward rectal
gland contents of the same and opposite sex was evaluated using Y-maze olfactometers. The system
consisted of a clear Plexiglas Y shape tube with one central arm (6.5 cm
the release chamber (5 cm
5 cm × 5 cm) was located, and two upwind lateral arms (12.5 cm
5 cm 5 cm) (see
× 4.5 cm × 5 cm) in which
×
×
4.5 cm × 5 cm), each of which was connected to a rectangular chamber (7.5 cm
×
×
Supplementary Materials, Figure S1). The Y-maze olfactometer was positioned horizontally on a
white table and a humidified and charcoal-filtered air stream was passed through the Y-maze at a
flow rate of 140
±
5 mL/min. The stimulus cartridge was prepared by crushing 15 rectal glands (male
or female) on a 1 cm2 filter paper (Advantec, Tokyo, Japan) inserted in a glass Pasteur pipette (145
mm long). The control cartridge was prepared using 1 cm2 filter paper inserted in the same type of

Molecules 2020, 25, 1275
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glass Pasteur pipette. One cartridge of each type was fitted to one of the Y-maze upwind arms using
a Tygon tube (Tygon formula E-3603, Sigma-Aldrich, St. Louis, MO, USA). An individual fly was
®
placed in the release chamber to acclimatise for 30 min before each experiment. The experiment was
carried out at noon in a controlled environment room, under the same conditions the flies were kept.
Each trial lasted 30 min. Once the two cartridges (stimulus and control) were connected to the upwind
arms, the system was allowed to equilibrate for two minutes and then the barriers of the two upwind
arms and the release chamber were removed. A choice was recorded when the fly reached one of the
two upwind arms and stayed there for at least one minute. Flies that did not make any choice, i.e.
remained in the release chamber, did not reach one of the two upwind arms, or did not stay there for
one minute, were excluded. For each treatment, 45–50 replicates from responsive flies were carried
out over multiple days with no more than 15 replicates on any day. The position (left or right) of the
stimulus and the control was reversed for every trial to minimise the positional effects. The flies used in
this experiment were obtained from multiple batches (two per week) and each fly was tested only once.
Freshly dissected rectal glands were used each day. Before each replicate, the Y-maze olfactometer was
washed with 5% Extran aqueous solution, rinsed with hot tap water, and air-dried. To compare the
number of flies choosing the stimulus over the control, a binomial test with the probability level of p <
0.05 was used.
3. Results
The GC-MS results showed that male and female B. frauenfeldi have different rectal gland and
volatile emission compositions. Females produced a more complex blend than did males (Figure 1).
A total of twenty-two compounds were identified including amides (
1 and 2), spiroacetals (3 and 4),
esters ( 10 12 15 18 21, and 22), and fatty acids (11 16, and 17). The identities of the twenty-one
5
–
,
–
,
–
,
compounds were confirmed by comparison with GC retention times and mass fragmentation patterns
of authentic samples.
Figure 1. Gas chromatogram of the rectal gland extract of B. frauenfeldi females. The numbered
peaks indicate detected compounds: N-(2-methylbutyl)acetamide (
(E,E)-2,8-dimethyl-1,7-dioxaspiro[ ]undecane ( ), (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane
), ethyl caprate ( ), methyl laurate ( ), ethyl laurate ( ), ethyl tridecanaote ( ), propyl laurate ( ), methyl
1), N-(3-methylbutyl)acetamide (2),
5,
5
3
(4
5
6
7
8
9
myristate (10), myristic acid (11), ethyl myristoleate (12), ethyl myristate (13), methyl palmitoleate (14),
methyl palmitate (15), palmitoleic acid (16), palmitic acid (17), ethyl palmitoleate (18), ethyl palmitate
(19), methyl elaidate (20), ethyl oleate (21), and ethyl elaidate (22).
Of the twenty-two compounds that were identified in female B. frauenfeldi, nine compounds were
detected in rectal gland extracts and headspace samples including N-(3-methylbutyl)acetamide (
(E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane ( ), (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane
), methyl myristate (10), ethyl myristoleate (12), ethyl myristate
13), and ethyl palmitoleate (18) (Table 1). The main compounds present in female gland extracts and
) and ethyl laureate ( ),
although they were found in higher proportions in the headspace samples (Table 1).
2),
3
(4), methyl laurate (6), ethyl laurate (7
(
headspace samples were (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (
3
7

Molecules 2020, 25, 1275
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Table 1. Percentage of compounds identified in chemical profiles for B. frauenfeldi. RT = retention time, KI = Kovats index, ND = not detected.
Females
Males
Rectal Gland (%)
Compound
Diagnostic Ions m/z (%)
RT
9.7
9.8
9.9
KI
Headspace (%)
Rectal Gland (%)
Headspace (%)
129 (M+, 5.2), 100 (62.2), 73 (β-cleavage/H
rearrangement, 76.4), 72 (M – C4H9, 100), 60
(CH3C(OH)NH+, 54.8)
N-(2-Methyl-butyl)acetamide
(1)
ND
<1
<1
ND
ND
ND
16.6
1133
1137
1147
129 (M+, 6.6), 114 (18.2), 86 (28.4), 73 (β-cleavage/H
rearrangement, 100), 72 (M – C4H9, 74.4), 60
(CH3C(OH)NH+, 32.6)
184 (M+, 9.7), 169 (2.1), 140 (17.8), 125 (9.7), 115
(CH3(C5H7O)=OH+, 98.1), 112 (CH3(C5H7O)=CH2,
100), 97 (75.4), 69 (33.3), 55 (31.2)
N-(3-Methylbutyl)acetamide
(2)
<1
ND
24.6
(E,E)-2,8-Dimethyl-1,7-
dioxaspiro[5.5]undecane (3)
53.9
20.1
198 (M+, 10.7), 169 (14.1), 140 (17.5), 129
(CH3CH2(C5H7O)=OH+, 52), 126
(E,E)-2-Ethyl-8-methyl-1,7-
dioxaspiro[5.5]undecane (4)
5.7
<1
75.4
70.3
11.3
1237
(CH3CH2(C5H7O)=CH2, 40.1), 115 CH3(C5H7O)=OH+,
94.2), 112 (CH3(C5H7O)=CH2, 100), 97 (66.5), 69 (43.5),
55 (49.1)
200 (M+, 1.7), 171 (4.2), 157 (19.5), 155 (M – OC2H5,
15.9), 115 (9.7), 101 (44.7), 88 (100), 73 (COOC2H5, 23.6),
70 (27.6)
214 (M+, 3.7), 183 (M – OCH3, 7.8), 171 (14.6), 143 (18.2),
87 (60), 74 (100), 59 (COOCH3, 8.4), 55 (22.8)
228 (M+, 4.3), 199 (4.7), 183 (M – OC2H5, 11.6), 157
(18.2), 101 (52.9), 88 (100), 73 (COOC2H5, 20.9), 70 (25.8),
61 (13.6), 55 (21.3)
Ethyl caprate (5)
Methyl laurate (6)
Ethyl laurate (7)
ND
2.1
<1
<1
ND
ND
ND
ND
ND
ND
13.5
15.1
15.9
1396
1524
1595
30.3
18.9
242 (M+, 4.5), 213 (11.9), 199 (15.6), 197 (M – OC2H5,
2.3), 157 (31.7), 101 (60.9), 88 (100), 73 (COOC2H5, 5.8),
57 (25.9), 55 (24.4)
Ethyl tridecanaote (8)
ND
<1
ND
ND
16.8
1667
242 (M+, 1.6), 201 (40.4), 199 (1.1), 183 (M – OC3H7,
36.5), 115 (26.7), 102 (29.7), 87 (COOC3H7, 11.2), 61 (100),
60 (34), 55 (30.4)
242 (M+, 6.6), 211 (M – OCH3, 6.3), 199 (16.2), 143 (25.6),
87 (64.4), 74 (100), 59 (COOCH3, 7.8), 55 (23.4)
228 (M+, 19.8), 185 (44.6), 171 (26.6), 143 (25.2), 129
(67.6), 115 (24.5), 97 (22.1), 87 (33.1), 85 (21.2), 83 (25.7),
73 (100), 69 (39.3), 60 (CH3COOH, 90.6), 57 (68), 55 (64)
Propyl laurate (9)
Methyl myristate (10)
Myristic acid (11)
ND
<1
<1
1.4
<1
ND
ND
ND
ND
ND
ND
17.1
17.4
17.8
1691
1727
1759
ND
254 (M+, 4.1), 209 (M – OC2H5, 13.9), 208 (M – C2H5OH,
14.9), 166 (28.8), 124 (23.7), 88 (46.3), 73 (COOC2H5,
16.6), 69 (52.1), 55 (100)
Ethyl myristoleate (12)
2.6
1.9
ND
ND
18.1
1785

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Table 1. Cont.
Females
Males
Compound
Diagnostic Ions m/z (%)
RT
KI
Headspace (%)
Rectal Gland (%)
Headspace (%)
Rectal Gland (%)
256 (M+, 7.1), 213 (13.8), 211 (M – OC2H5, 8.16), 157
(21.9), 101 (53.8), 88 (100), 73 (COOC2H5, 17.8), 70 (22.1),
55 (20.1)
Ethyl myristate (13)
1.9
14.6
ND
ND
18.2
1795
268 (M+, 5.1), 237 (M – OCH3, 14.2), 236 (M – CH3OH,
18.5), 194 (17.9), 152 (24.1), 96 (51.3), 74 (52.3), 59
(COOCH3, 17.1), 55 (100)
270 (M+, 12.5), 227 (14.8), 143 (23.6), 87 (68.2), 74 (100),
69 (12.5), 59 (COOCH3, 7.2), 55 (24.8)
254 (M+, 2.2), 236 (13.6), 152 (9.2), 111 (23.8), 98 (33.8), 97
(50.3), 96 (35.2), 83 (56.4), 73 (15.3), 69 (73.7), 60
(CH3COOH, 10), 57 (24.8), 55 (100)
Methyl palmitoleate (14)
Methyl palmitate (15)
Palmitoleic acid (16)
ND
ND
ND
2.5
<1
5.5
ND
ND
ND
ND
ND
4.4
19.3
19.5
19.7
1909
1928
1825
256 (M+, 38.1), 227 (9.9), 213 (M – COOH, 31.3), 185
(26.9), 157 (31.4), 129 (61.8), 115 (26.5), 97 (33.2), 87 (36.7),
85 (37), 83 (39), 73 (100), 69 (45.9), 60 (CH3COOH, 84.8),
57 (88.9), 55 (75.4)
282 (M+, 2.9), 237 (M – OC2H5, 19.1), 236 (M – C2H5OH,
21.3), 194 (23.2), 152 (28.6), 88 (57.3), 73 (COOC2H5,
16.8), 69 (68.7), 55 (100)
Palmitic acid (17)
ND
2.5
3.1
ND
ND
3.9
19.9
20.0
1962
1977
Ethyl palmitoleate (18)
16.1
ND
284 (M+, 11.2), 255 (4.1), 241 (13.2), 239 (M – OC2H5,
7.5), 157 (21.3), 101 (57.5), 88 (100), 73 (COOC2H5, 16.1)
296 (M+, 5.3), 265 (M – OCH3, 17.8), 264 (26.7), 222
(16.9), 152 (13.6), 97 (62.2), 74 (47.5), 69 (66.2), 55 (100)
Ethyl palmitate (19)
Methyl elaidate (20)
ND
ND
5.3
ND
ND
ND
ND
20.2
21.2
1995
2102
<1
310 (M+, 1.2), 265 (M – OC2H5, 8.8), 264 (M – C2H5OH,
16.9), 222 (5.4), 123 (13.6), 110 (22.8), 97 (59.7), 88 (54.1),
83 (62.9), 73 (COOC2H5, 15.1), 69 (72.1), 55 (100)
310 (M+, 7.9), 265 (M – OC2H5, 24.5), 264 (M – C2H5OH,
31.7), 222 (22.1), 180 (20.4), 110 (31.4), 97 (65.5), 88 (57.9),
83 (63.4), 73 (COOC2H5, 15.2), 69 (68.4), 55 (100)
Ethyl oleate (21)
ND
ND
6.4
1.9
ND
ND
4.8
21.6
21.8
2144
2172
Ethyl elaidate (22)
ND

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The GC-MS analysis of male rectal gland extracts showed a blend of six compounds
(Figure 2), being (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane ( ) as the main compound and
(E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane ( ) as the second major compound, representing 70%
4
3
and 17% of the blend, respectively. Male headspace samples showed only (E,E)-2-ethyl-8-methyl-1,7-
dioxaspiro[5.5]undecane and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, in a similar ratio to that
detected in the male rectal gland samples.
Figure 2. Typical Gas chromatogram of rectal gland extract of B. frauenfeldi males. The
numbered peaks indicate detected compounds: (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (
3),
(E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane (4), palmitoleic acid (16), palmitic acid (17), and
ethyl oleate (21).
3.1. Electrophysiological Responses
The analysis by GC-EAD of the male rectal gland samples showed that female and male antennae
responded consistently to the main spiroacetal, (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane
(
(
3
4
). The female antennae also responded to (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane
) from the male rectal gland, while the male antennae did not. The female and male palps
responded to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (3) and (E,E)-2-ethyl-8-methyl-1,7-
dioxaspiro[5.5]undecane (4) (Figure 3A).
Figure 3. Flame ionization detector (FID) response and electrophysiological responses of antennae
(EAD) and maxillary palps (EPD) using Bactrocera frauenfeldi males and females to ( ) rectal gland
extracts from conspecific males and ( ) rectal gland extracts from conspecific females. The numbered
A
B
peaks indicate EAD- and EPD-active compounds: (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (
(E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane (4), ethyl caprate (5), and methyl laurate (6).
3),

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In contrast, GC-EAD analysis of female rectal gland samples revealed that
(E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane elicited an antennal response in both
males and females. The female antennae responded to two more compounds from the
female rectal gland including (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane ( ) and ethyl
caprate ( ). GC-EPD analysis of the female rectal gland samples showed that the female
and male maxillary palps responded to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane ( ) and
) was only detected by the
(3)
4
5
3
(E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane (4). Methyl laurate (6
female palps (Figure 3B).
3.2. Y-maze Bioassays
Sexually mature B. frauenfeldi females significantly preferred the upwind arm containing male
rectal glands over the control (p = 0.01). In contrast, B. frauenfeldi males did not show a significant
preference for female rectal glands over the control (p = 0.1). Neither females nor males exhibited
significant preferences when the rectal gland content of the same sex was presented (p = 0.06 and p =
0.1, respectively) (Figure 4).
Figure 4. Response of sexually mature virgin Bactrocera frauenfeldi males and females to rectal gland
volatiles of the same and opposite sex, vs. control (clean filter paper) in Y-maze bioassays. * significantly
different at 0.01 level, ns not significantly different, n total number of responded flies.
4. Discussion
We report here the first identification, electrophysiological detection, and behavioural evaluation
of rectal gland volatiles produced by B. frauenfeldi males and females. Our data show that females
of this species produced and emitted a greater diversity of compounds than males. The compounds
included aliphatic amides, spiroacetals, and saturated and unsaturated acids and esters. The two
amides, N-(2-methylbutyl)acetamide (
1) and N-(3-methylbutyl)acetamide (
2), found in this study
have been previously reported in rectal glands of other species, including B. tryoni [31
,58], Z. cucumis
(French) [34], B. dorsalis (Hendel), and Z. cucurbitae (Coquillett) [45]. N-(3-Methylbutyl)acetamide is one
of the major components in the rectal glands of B. tryoni males. The two spiroacetals that were found
in B. frauenfeldi have also been reported in other Bactrocera species, and closely related Zeugodacus. For
instance, EE, EZ, and ZZ isomers of 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane have been reported
in the rectal glands of Z. cucumis [33]. (E,E)-2,8-Dimethyl-1,7-dioxaspiro[5.5]undecane (
also found in the rectal glands of B. dorsalis, B. nigrotibialis (Perkins), B. albistrigata (Meijere), B. jarvisi
(Tryon), B. kirki (Froggatt), B. kraussi (Hardy), B. musae (Tryon), and B. tryoni [33 34 45 58 61]. This
3) have been
,
, , –
compound was also found in both sexes of B. frauenfeldi, although it was less abundant in males. The
most abundant compound in males was (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane. The
spiroacetal (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane has been previously reported in the
rectal glands of male B. nigrotibialis (Perkins), B. halfordiae (Tryon), B. dorsalis (Hendel), B. kirki (Froggatt),
B. latifrons (Hendel), and B. occipitalis (Bezzi) and in female B. tryoni (Froggatt) and B. musae [
In addition to the compounds found in males, the female rectal gland and headspace extracts included
saturated and unsaturated acids and esters. The compounds, ethyl caprate ( ), methyl laurate ( ),
4,58,61–63].
5
6

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ethyl laurate (
7
), ethyl tridecanaote (8), propyl laurate (
9), methyl myristate (10), myristic acid (11),
ethyl myristoleate (12), ethyl myristate (13), methyl palmitoleate (14), methyl palmitate (15), ethyl
palmitoleate (18), ethyl palmitate (19), methyl elaidate (20), and ethyl elaidate (22) were female-specific.
Nine of the saturated and unsaturated esters in B. frauenfeldi have also been reported in B. oleae, with
ethyl myristate and ethyl palmitate as the major compounds in both species [49]. B. musae rectal glands
were also reported to contain twelve of the esters found in female B. frauenfeldi with ethyl laurate, ethyl
myristate, and ethyl palmitate as the most abundant components [61]. Ethyl caprate, which was EAG
active for females of B. frauenfeldi, has been found to attract both males and females of B. oleae [49].
The other electrophysiologically active ester in this study, methyl laurate, has not been reported as
an attractant in any other species, but the similar saturated methyl ester, methyl palmitate, has been
reported to attract both male and female B. oleae [49]. Many of the saturated/unsaturated acids and
esters that we found in B. frauenfeldi females are also common in females of other Bactrocera species,
although ratios vary amongst species.
The differences between the composition of rectal glands and volatile emissions observed in
this study are not likely due to sampling and/or sensitivity of the instrument because allowing for
those factors, the ratios in the rectal gland samples would be matched in the headspace collections.
This is not the case. In female rectal glands compounds
1%) but only compound was detected in the headspace samples. Similarly, compounds 16 and 19
were more abundant than compound in female rectal glands (both > 5%) but were not detected in
5 and 6 have similar relative abundance (<
6
6
headspace samples. For the compounds with longer chain length, and hence lower volatility including
the C14, C16, and C18 esters, compared to the C12 ester, it is likely that the relative abundance of these
compounds is lower in headspace than rectal glands. The differences between the composition of rectal
glands and volatile emissions could be due to the disproportionate release of some compounds.
The proportion of compounds in a blend can be critical for triggering behavioural responses,
and the differences might be responsible for sex-specific behavioural responses. For example, in
closely related moth species, pheromone blends often contain the same compounds but in different
ratios [64
instance, the spiroacetals
,
65]. In the present study we found sex differences in the proportion of some compounds. For
and are present in very different ratios in the rectal gland of males and
3
4
females; 19:81 in males vs. 99:1 in females. If the presence of the compounds was solely responsible for
behavioural response, females should have had a preference for female glands in Y-maze bioassays.
The fact that this was not the case suggests natural proportions of the compounds may be important in
triggering sex-specific behavioural responses.
Male production of sex-attracting pheromones has been reported in diverse tephritid fruit
flies [25
pheromone components including (Z)-non-3-en-1-ol and (3Z,6Z)-nona-3,6-dien-1-ol, anastrephin,
and epianastrephin, which attract female conspecifics [25 66]. The male Mediterranean fruit fly,
Ceratitis capitata (Wiedemann), releases a cyclic imine, l-pyrroline, that is highly attractive to virgin
females [34 67]. In B. tryoni, male glandular blends include the amides, N-3-methylbutylpropanamide,
,34,66]. For example, the male Caribbean fruit fly, Anastrepha sunspensa (Loew), releases volatile
,
,
N-3-methylbutylacetamide, N-(3-methylbutyl)-2-methylpropanamide, N-2-methylbutylpropanamide,
N-2-methylbutylacetamide, and N-(2-methylbutyl)-2-methylpropanamide, which are thought to
function as short-range stimulants for females [31,34]. Our Y-maze olfactometer results showed
that female B. frauenfeldi are attracted to male gland odour, whereas males did not exhibit
a significant preference for male or female gland odours during the period of peak mating
activity. This suggests that a specific compound or specific compounds produced in the rectal
gland of B. frauenfeldi males elicits attraction of females. Our GC-EAD/EPD results showed that
(E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane elicits antennal and palpal responses in both males and
females. Although (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane elicits an antennal response only
in females, it was detected by palps in both males and females. Our findings suggest that in B. frauenfeldi
males are the main sex pheromone producer and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and
(E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane are sex pheromone candidates. This chemical

Molecules 2020, 25, 1275
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information about the sexual communication of B. frauenfeldi provides a valuable foundation for the
synthesis and development of new nature-inspired attractants for the control of this pest species.
Supplementary Materials:
The supplementary materials are available online.
Synthesis of
N-(2-methylbutyl)acetamide, N-(3-methylbutyl)acetamide, 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, ethyl
palmitoleate, and ethyl elaidate. Figure S1. Y-maze apparatus used in this study.
Author Contributions: Conceptualization, S.N., P.T., I.J. and J.J.; Investigations, S.N., J.P., D.H., S.J.P. and V.M.A.;
Data analysis, S.N.; Writing—Original draft preparation, S.N.; Writing—Review and editing, P.T., J.J., I.J., J.P. and
S.J.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the Australian Research Council Industrial Transformation Training
Centre (ITTC) for Fruit Fly Biosecurity Innovation (Project IC50100026), funded by the Australian Government.
Acknowledgments: We gratefully acknowledge the Queensland Department of Agriculture and Fisheries (QDAF),
especially Sybilla Oczkowicz and Peter Leach, for providing B. frauenfeldi from their colonies for this research.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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