1-Octanol emitted by Oecophylla smaragdina weaver ants repels and deters oviposition in Queensland fruit fly

Abstract

Humans have used weaver ants, Oecophylla smaragdina, as biological control agents to control insect pests in orchards for many centuries. Over recent decades, the effectiveness of weaver ants as biological control agents has been attributed in part to deterrent and oviposition inhibiting effects of kairomones produced by the ants, but the chemical identity of these kairomones has remained unknown. We have identified the kairomone responsible for deterrence and oviposition inhibition by O. smaragdina, providing a significant advance in understanding the chemical basis of their predator/prey interactions. Olfactometer assays with extracts from weaver ants demonstrated headspace volatiles to be highly repellent to Queensland fruit fly, Bactrocera tryoni. Using electrophysiology and bioassays, we demonstrate that this repellence is induced by a single compound, 1-octanol. Of 16 compounds identified in O. smaragdina headspace, only 1-octanol evoked an electrophysiological response from B. tryoni antennae. Flies had greatly reduced oviposition and spent significantly less time in an olfactometer arm in the presence of 1-octanol or a synthetic blend of headspace volatiles containing 1-octanol than in the presence of a synthetic blend of headspace volatiles without 1-octanol, or clean air. Taken together, our results demonstrate that 1-octanol is the functional kairomone component of O. smaragdina headspace that explains repellence and oviposition deterrence, and is hence an important contributor to the effectiveness of these ants as biological control agents.

Full text

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1‑Octanol emitted by Oecophylla
smaragdina weaver ants
repels and deters oviposition
in Queensland fruit y
Vivek Kempraj*, Soo Jean Park, Donald N. S. Cameron & Phillip W. Taylor
Humans have used weaver ants, Oecophylla smaragdina, as biological control agents to control
insect pests in orchards for many centuries. Over recent decades, the eectiveness of weaver ants as
biological control agents has been attributed in part to deterrent and oviposition inhibiting eects
of kairomones produced by the ants, but the chemical identity of these kairomones has remained
unknown. We have identied the kairomone responsible for deterrence and oviposition inhibition by
O. smaragdina, providing a signicant advance in understanding the chemical basis of their predator/
prey interactions. Olfactometer assays with extracts from weaver ants demonstrated headspace
volatiles to be highly repellent to Queensland fruit y, Bactrocera tryoni. Using electrophysiology
and bioassays, we demonstrate that this repellence is induced by a single compound, 1‑octanol. Of
16 compounds identied in O. smaragdina headspace, only 1‑octanol evoked an electrophysiological
response from B. tryoni antennae. Flies had greatly reduced oviposition and spent signicantly less
time in an olfactometer arm in the presence of 1‑octanol or a synthetic blend of headspace volatiles
containing 1‑octanol than in the presence of a synthetic blend of headspace volatiles without
1‑octanol, or clean air. Taken together, our results demonstrate that 1‑octanol is the functional
kairomone component of O. smaragdina headspace that explains repellence and oviposition
deterrence, and is hence an important contributor to the eectiveness of these ants as biological
control agents.
Weaver ants are voracious predators and have been used as biological control agents for many centuries to
control insect pests in Asia (Oecophylla smaragdina) and in Africa (Oecophylla longinoda). A common practice
in Asia involves establishing a nest on one tree and then connecting it to adjacent trees with bamboo poles,
thus enabling the movement of ants throughout the orchard to forage1,2. Records of this practice can be found
in the 1726 Imperial Encyclopedia of the Ching dynasty and in a regional botany work written by Ji Han in
3042. Weaver ants have been found to be eective in controlling insect pests of mango3–8, cashew9,10, citrus11,12,
coconut13 and cocoa14,15. While direct eects of predation by weaver ants are certainly important, recent studies
have highlighted that repellence and oviposition deterrence induced by chemical emissions (kairomones) from
the ants are also important elements of crop protection conferred by weaver ants5,16. Unidentied weaver ant-
produced kairomones have been found to inhibit oviposition by fruit y pests including Bactrocera jarvisi5, B.
dorsalis and Ceratitis cosyra17,18. In the eld, damage to mango fruits by B. jarvisi is decreased in the presence
of O. smaragdina5. When presented with O. longinoda-exposed and unexposed mango fruits in the absence of
ants, B. dorsalis and C. cosyra land less oen on ant-exposed fruits and if they do land tend to depart quickly
and fail to oviposit17. Volatile olfactory cues from O. smaragdina induce increases in motility (velocity, active
time and distance moved) and reductions in foraging, oviposition and mating propensity in the Queensland
fruit y Bactrocera tryoni19.
While numerous studies have demonstrated fruit y responses to kairomones from weaver ants, and have
strongly implicated such kairomones as an important element of biological control5,17,18, the specic compounds
responsible remain unknown. Weaver ants are known to emit and deposit a diversity of compounds, including
hydrocarbons, esters, fatty acids, terpenes, and alcohols. Hydrocarbons make up ~ 90% of the compounds emit-
ted by O. smaragdina with n-undecane being a highly emitted compound (~ 45%)20. Identifying kairomones
that mediate responses of prey to predators can provide valuable insights to subtle aspects of predator–prey
interactions and can also provide insights into how kairomones aect food webs21,22. Furthermore, when a
OPEN
Applied BioSciences, Macquarie University, Sydney, NSW, Australia. *email: vivek.kempraj@gmail.com
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predator-released kairomone repels or deters oviposition in a pest species, it can have a signicant impact on
pest populations23,24 and may even be developed as an eective pest management tool. In the present study, we
identify a single compound from the headspace volatiles emitted by O. smaragdina that is detected by antennae,
induces repellence, and deters oviposition in B. tryoni. is knowledge signicantly advances understanding of
predator–prey interactions between weaver ants and fruit ies, and lays the foundations for the development of
biologically inspired repellents that could oer a new tool for non-insecticidal, safe, management of economi-
cally important fruit ies.
Results
Olfactometer screening of extracts and volatile emissions of O. smaragdina revealed that only head extract and
headspace volatiles repel B. tryoni (Fig.1). Male and female ies spent signicantly more time in the control
arm of olfactometers (Male: 5.129 ± 0.65min (mean ± s.e.m), t = 2.383, df = 19, P = 0.02; Female: 5.885 ± 0.73min,
t = 2.249, df = 19, P = 0.03; Fig.1e) than in the treatment arm containing head extract (Male: 2.916 ± 0.45min;
Female: 3.459 ± 0.54min; Fig. 1e). A similar, but a stronger repellence was observed in response to head-
space volatiles of O. smaragdina. Male and female ies spent signicantly more time in the control arm (Male:
7.620 ± 0.34min; t = 9.839; df = 19; P < 0.001; Female: 6.713 ± 0.73min, t = 9.370, df = 19, P < 0.001; Fig.1f ) than
in the treatment arm containing headspace volatiles (Male: 1.498 ± 0.36min; Female: 0.3755 ± 0.14min; Fig.1f).
Headspace volatiles were explored further by Gas Chromatography-Electroantennographic Detection (GC-
EAD) to identify compounds that might be responsible for repellence of B. tryoni. Both male and female ies
responded very consistently to a single compound in the headspace volatiles, and from Gas chromatograph
mass spectrometry (GC–MS) analysis the electrophysiologically active compound was found to be 1-octanol
(Fig.2). Although we observed a single compound to be electrophysiologically active on antennae, which are
thought to mediate long range olfactory responses, B. tryoni do have olfactory receptors on other body parts
(e.g., maxillary palps)25 and so the possibility remained that other compounds may be detected by organs other
than those on antennae and be responsible for repellence. To conrm the functional eect of 1-octanol as a
repellent, we prepared two synthetic blends of headspace volatiles, one that contained all components includ-
ing 1-octanol (BL+OL) and one that contained all components except 1-octanol (BL−OL). In olfactometer assays,
male and female ies were not repelled by the blend BL−OL, spending similar amounts of time in the control arm
(Male: 4.848 ± 0.53min; t = 0.4238; df = 19; P = 0.67; Female: 4.459 ± 0.28min, t = 1.106, df = 19, P = 0.28; Fig.3a)
and the treatment arm (Male: 4.481 ± 0.55min; Female: 3.922 ± 0.35min). However, when presented with the
blend BL+OL, both male and female ies spent signicantly more time in the control arm (Male: 5.562 ± 0.64min;
t = 4.406; df = 19; P < 0.001; Female: 5.136 ± 0.61min, t = 7.635, df = 19, P < 0.001; Fig.3b) than in the treatment
arm (Male: 1.669 ± 0.47min; Female: 0.366 ± 0.12min). Next, 1-octanol alone was evaluated for its deterrence
of B. tryoni to test whether ies responded to 1-octanol outside the context of ant volatiles. Male and female ies
spent signicantly more time in the control arm (Male: 7.738 ± 0.59min; t = 12.110, df = 19, P < 0.001; Female:
6.354 ± 0.55min; t = 8.299, df = 19, P < 0.001; Fig.3c) than in the treatment arm containing 1-octanol (Male:
0.7765 ± 0.18min; Female: 1.424 ± 0.17min).
In oviposition assays, gravid females laid signicantly more eggs into control agarose plates (113.2 ± 14.05
eggs; mean ± s.e.m) and BL−OL plates (containing all headspace components except 1-octanol) (71.7 ± 6.33 eggs)
than into BL+OL plates (containing all headspace components including 1-octanol) (2.5 ± 0.73 eggs) or OL plates
(containing only 1-octanol) (2.2 ± 0.59 eggs)(F3, 36 = 50.2; P < 0.001; Fig. 4). e presence of 1-octanol almost
completely inhibited oviposition, but it is important also to note that the eect was by contact or short range
olfaction because such inhibition did not carry over to other plates in the same cage. Taken together, our results
demonstrate that 1-octanol is responsible for kairomonal eects of repellence and oviposition deterrence in B.
tryoni that are exposed to olfactory cues from O. smaragdina weaver ants.
Discussion
Kairomones released by predators can signicantly inuence prey species behaviour and life history26–29. Despite
numerous studies demonstrating kairomonal eects of olfactory cues released by predators21,28,30, there are sur-
prisingly few studies providing chemical characterisation of such kairomones31–33.
Although olfactory cues produced by weaver ants (O. smaragdina in Asia and Australia and O. longinoda
in Africa) have been known to have a strong repellent eect on fruit ies3–8,11,12, the present study is the rst to
chemically identify and demonstrate eects of kairomonal components. Active compounds in the headspace
appear to originate in the head of O. smaragdina. 1-octanol was found to be the only one of 16 compounds in the
headspace to elicit electrophysiological responses in B. tryoni antennae, and the kairomonal function of 1-octanol
as a repellent and oviposition deterrent was demonstrated using bioassays that presented headspace blends with
and without 1-octanol. GC–MS analysis of the body extracts and volatile emissions revealed 1-octanol in the head
extract and headspace volatiles. 1-Octanol was previously reported from head extracts and mandibular glands
of O. smaragdina20,34,35, and also in headspace20. Previous studies have found that 1-octanol in honeybee alarm
pheromone repels the parasitic mite, Varroa jacobsoni36. Octanol (unspecied conguration) has been reported
as a minor component in the endocrine secretions of cockroaches, although no function has been identied37.
1-Octanol in essential oils of plants has been reported as a biting deterrent in the mosquito Aedes aegypti38 and
as an oviposition deterrent in the Asian corn borer, Ostrinia furnacalis39. A recent study has shown 1-octanol to
be a major component of the alarm pheromones in a mammal, the Bank vole Myodes glareolus40. However, the
function of 1-octanol in O. smaragdina is currently unknown. Given the alarm pheromone function of 1-octanol
in honeybees, also a social hymenopteran, a similar function warrants investigation in O. smaragdina.
Gravid B. tryoni rely on fruit volatiles when detecting and choosing fruits for oviposition41–43. We used
γ-octalactone, a strong oviposition stimulant of B. tryoni44 and a short-range attractant in some tephritid fruit
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ies45, to establish a high baseline of oviposition in order to demonstrate the substantial inhibitory eects of
1-octanol. In oviposition assays, 1-octanol in the presence or absence of other O. smaragdina headspace com-
ponents over-rode the oviposition-stimulating eect of γ-octalactone resulting in very low levels of oviposition.
Identifying and characterizing predator-released kairomones paves the way for more detailed studies of how
prey behaviour and food web structure can be aected by such public information. Identifying the predator-
released kairomones that inuence oviposition by B. tryoni and other fruit ies also provides foundations for the
development of new, sustainable, pest management tools. Kairomone-based repellents and oviposition deterrents,
such as 1-octanol, could potentially be exploited to protect crops and reduce reliance on environmentally harmful
insecticides. In addition to eects on gravid female B. tryoni, 1-octanol was found to be repellent to male ies
and could potentially contribute to reduced mating in pest populations.
Figure1. Behavioural response of male and female B. tryoni to O. smaragdina body extracts and volatiles. (A)
cuticular compounds; CH, (B) Dufour gland; DG, (C) poison gland; PG, (D) trail extract; TR (E) head extract;
HD and (F) headspace volatiles; HS were tested. Only head extract and headspace volatiles signicantly repelled
ies. Male and female ies spent signicantly more time in control arm (Yeast Hydrolysate; YH) than the
treatment arm (Yeast Hydrolysate + Head extract or Yeast Hydrolysate + Headspace voltiles). Error bar represent
s.e.m. Signicant dierence was analysed by paired t-test (see Results).
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Materials and methods
Insects. Bactrocera tryoni were obtained from a colony that originated from infested fruit collected in central
coastal New South Wales and had been maintained in a controlled environment laboratory (25 ± 0.5°C, 65 ± 5%
RH, photoperiod of 11.5:0.5:11.5:0.5 light: dusk: dark: dawn) at Macquarie University for 32 generations. From
emergence, adult ies were fed yeast hydrolysate, sugar and water adlibitum and were used in experiments when
10 to 15days old, when sexually mature46. Major workers of O. smaragdina were collected from ve dierent
colonies in the vicinity of Mareeba Research Facility, Department of Agriculture and Fisheries, QLD, Australia
(17.00724°S, 145.42984°E).
Chemicals. Authentic standards of 1-hexanol, decane,p-cymene, D-limonene, γ-terpinene, 1-octanol, dihy-
dromyrcenol, undecane, nonanal, dodecane, tridecane, 1-tetradecene, tetradecane, pentadecane, hexadecane,
heptadecane (all known components of emissions produced by O. smaragdina)20 and hexane were purchased
from Sigma-Aldrich. All chemicals were of analytical grade (≥ 98% purity) and were used without further puri-
cation.
Collection of body & gland extracts, volatile emissions, and trail extracts. Cuticular com-
pounds, head extracts, gland extracts (Dufour and poison glands), headspace volatiles and trail extracts of O.
smaragdina were collected as described by Kempraj etal.20. For cuticular compounds, individual ants (n = 100)
were dipped in 10mL of hexane for 10s. For head extracts, heads of ants (n = 10) were removed with dissection
scissors and immediately placed in 1.5mL of hexane in a glass vial for 24h. e extraction time for cuticular
compounds and head extract was crucial in achieving dierentiation in the compounds extracted. e extended
extraction time for head extracts enabled extraction of glandular compounds present in the head (mandibular
glands, intramandibular glands, propharyngeal and postpharyngeal glands), whereas the short extraction time
for cuticular compounds was enough to extract compounds on the cuticle without signicant extractions from
glands. For gland extracts, Dufour and poison glands were dissected from the abdomen and remnant tissues
were carefully removed using ne forceps. Clean glands (n = 10) were immediately placed into 1.5mL of hexane
in a glass vial. Glands were extracted by standing the vial at room temperature for 24h. Headspace volatiles
present in the air surrounding the ants was collected using an air entrainment system. Ten ants were placed in a
cylindrical glass chamber (120mL capacity) with an inlet and outlet and were allowed to acclimatize for 30min
prior to collection of volatiles. A charcoal lter was connected to the inlet (4mm ID) of the glass chamber using
Tygon tubing (E-3603). e outlet of the glass chamber was connected to a Tenax tube (50mg, Scientic Instru-
ment Services Inc, Tenax-GR Mesh 60/80, packed in 6 × 50mm glass tubes) tted to a screw cap with O-ring.
Nine chambers containing ants and one empty control chamber were set up for each run. Headspace volatiles
were adsorbed onto Tenax at a ow rate of 0.5 L/min for 30min by pulling air from the outlet using a pump
(KNF Pumps, Model no. NMP850.1.2KNDCB, Switzerland). For trail extracts, we found a metal fence that
served as a regular path to transport food and other materials to the nest by O. smaragdina. Prior to collection,
the section of metal fence (ca.3m) that the ants used to commute was rinsed with acetone (100mL) to remove
pre-existing trail chemicals. e ants were allowed to make a trail on the rinsed section of the mesh for 24h.
Between 2 and 4pm Standard Australian Time (when weaver ants are highly active) the metal wire was rinsed,
section by section, with a total of 100mL hexane into a 500mL glass beaker. e trail wash was concentrated
under a gentle stream of clean air down to approximately 10mL. All collections were at least ten replicates and
stored at 4°C until further processing. Samples of body extracts and gland extracts were treated with a drying
agent (sodium sulfate) and by gravity ltration with a glass wool plugged Pasteur pipette to remove water and
debris. Samples free from water and debris were concentrated under a gentle stream of nitrogen gas. Cuticular
compound samples were concentrated to 1mL while Dufour’s gland, poison gland and head samples were con-
centrated to 0.5mL. Trail samples were ltered to remove solid matter and concentrated to 1mL under a gentle
stream of nitrogen gas. Headspace volatile samples did not require further processing. All processed samples
were stored at − 20°C until analysis.
Figure2. Representative GC-EAD recording of male and female B. tryoni response to headspace volatiles of
O. smaragdina. In both male and female ies the FID peak marked as ‘1-octanol’ was the only compound that
elicited consistent response.
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Gas chromatography mass spectrometry (GC–MS) analysis. GC–MS analysis of all samples were
carried out on a Shimadzu GC–MS TQ8030 spectrometer equipped with a split/splitless injector and SH RTX-
5MS (30m × 0.25mm, 0.25µm lm) fused silica capillary column. Carrier gas was helium (99.999%) at a ow
rate of 1mL/min. An aliquot of 1 µL was injected in splitless mode, with injector temperature set at 270°C. e
temperature program was as follows: 50°C for 1min, increased to 280°C at 10°C min−1 and increased to 300°C
at 5°C min−1. e ion source and transfer line temperatures were 200°C and 290°C respectively. e ionization
method was electron impact at a voltage of 70eV. Spectra were obtained over a mass range of m/z 45–650. For
the identication of compounds, mass fragmentation patterns were compared with NIST library (NIST17-1,
NIST17-2, NIST17s) and Kovats retention indices were compared with literature values. e identities of the
compounds were conrmed by comparing retention index and fragmentation patterns of each compound with
authentic standards.
Figure3. Behavioural response of male and female B. tryoni to synthetic blends with (BL+OL) or without
(BL−OL) 1-octanol, and 1-octanol alone (OL). In behavioral assays using synthetic blend of headspace voltiles
without 1- octanol, male and female ies spent similar time in control (YH; Yeast hydrolysate) and treatment
(YH + BL−OL) arms. However, in behavioural assays using a synthetic blend of headspace volatiles with 1-octanol
or 1-octanol alone, male and female ies spent signicantly (P < 0.001) more time in control (YH) than
treatment (YH + BL+OL) arms. Error bar represent s.e.m. Signicant dierence was analysed by paired t-test (see
Results).
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Electrophysiology. Coupled Gas Chromatography-Electroantennographic Detection (GC-EAD) record-
ings were made using Ag-glass microelectrodes lled with electroconductive gel (Spectra 360, Parker Laborato-
ries Inc., USA) (n = 6). A male or gravid femaleof B. tryoni was subdued by chilling, and the head was separated
from the body using a microscalpel. e base of the head was then xed to the tip of the gel-lled indierent
electrode. e tip of an antenna was placed in contact with the recording electrode and was slightly inserted into
the gel to stabilize the antenna. e signals were passed through a high impedance amplier (UN-06, Syntech,
Hilversum, e Netherlands). Headspace samples were tested by injecting of 1µl of sample into the GC column.
Euent from the GC column was simultaneously directed to the antennal preparation and the GC detector
at a split ratio of 1.5:1, respectively. Separation of compounds was achieved on a Agilent GC 7890B equipped
with a split/splitless injector and a ame ionization detector (FID), using an HP-5 column (30m, 0.32mm ID,
0.25μm lm, Agilent, CA, US). e carrier gas was hydrogen (99.999%) (BOC, North Ryde, NSW, Australia) at
a ow rate of 3.0mL/min. e injector temperature was 270°C. e oven temperature was maintained at 45°C
for 2min, and then increased to 250°C at 10°C min−1. e outputs from the EAG amplier and the FID were
monitored simultaneously by GcEad soware ver. 1.2.5 (Syntech, Kirchzarten, Germany). Peaks eluting from
the GC column were judged to be active if they elicited EAD activity in six or more of the ten coupled runs. e
identities of FID peaks were conrmed by GC–MS (Shimadzu TQ8030) operating at the same GC conditions
with the same type of column (5% diphenyl and 95% dimethyl polysiloxane).
Preparation of synthetic blends of headspace volatiles. GC–MS results of weaver ant headspace
samples guided the preparation of two synthetic blends. e 16 identied headspace compounds20 were used to
prepare two synthetic blends that matched the relative abundance of compounds in the natural blend. One syn-
thetic blend contained all the headspace components including 1-octanol (BL+OL) (BL = Blend; OL = 1-octanol),
while the other synthetic blend contained all the headspace components except 1-octanol (BL−OL). Stock solu-
tions of the headspace compounds with a concentration range of 5.0–10.0mg/mL in hexane were prepared
in 10mL volumetric asks. e stock solutions were run through GC to obtain response factors for the given
concentration. e response factor of undecane was used as a reference to adjust the volumes of each compound
added to the synthetic blend. e calculated volumes of the compounds were added to a 10mL volumetric ask.
e ask was lled with hexane to the mark and inverted several times to mix the blend well. e synthetic blend
was run through GC to conrm if the relative gas chromatographic (GC) intensities of the compounds were
consistent with that in the natural headspace volatile extract. Preparing a synthetic blend and comparing GC
intensities were repeated several times until the relative GC intensities were consistent with that in the natural
headspace volatile extract. e concentration of undecane, the reference compound, was arbitrary each time but
in a range of 10.0 to 15.0μg/mL. e GC conditions used in this process were the same as the above GC–MS
analysis, except that 1μl of sample was injected at split mode (a ratio of 1:60).
Olfactometer bioassays. An acrylic four-arm olfactometer (120mm diameter; see Fig.S1) was used to
assess behavioural responses of male and female B.tryoni to extracts of cuticle, Dufour gland, Poison gland,
Trail and head and volatile emissions of weaver ants as well as synthetic blends (BL+OL, BL−OL) or 1-octanol (OL)
alone. Prior to each experiment, olfactometers were washed with a non-ionic detergent solution, rinsed with
ethanol and distilled water, and le to air dry. Experiments were conducted in a controlled environment room
(25 ± 2°C, 60% RH). To provide traction for the walking insects, lter paper (Whatmann No. 1, 12cm diameter)
was placed on the oor of the central area. e room was illuminated from above by uniform lighting from white
LED lights. Individual ies(10–15days old, without access to food over the preceding 24h, but with access to
Figure4. 1-octanol inhibited oviposition by gravid B. tryoni females. Gravid females were presented with
agarose plates containing oviposition stimulant (OS) alone (control), agarose plates containing OS + synthetic
blend of headspace volatiles excluding 1-octanol (BL−OL), agarose plates containing OS + synthetic blend of
headspace volatiles including 1-octanol (BL+OL) and agarose plates containing OS + 1-octanol (OL). Signicantly
more eggs were laid into control and BL−OL than into BL+OL and OL. Error bars represent s.e.m. Signicant
dierence is denoted by dierent letters (ANOVA followed by Tukey’s test; P < 0.001; n = 10; see Results).
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water) were introduced to the olfactometer through a hole in the oor. Each y was given 5min to acclimatize
in the olfactometer, aer which the experiment was run for 10min. e olfactometer was rotated 90° aer each
replicate to eliminate any directional bias. Air was drawn through the central hole at 200ml min−1and subse-
quently exhausted into the room. e central arena of the olfactometer was divided into four discrete odour
elds corresponding to each of four inlet arms. A choice test was performed that used two opposite arms and
the other two arms were closed and were not used in the test. One arm was for treatment and the opposite arm
was control. Test samples (extracts, volatile emissions, BL+OL, BL−OL or OL-1.17% v/v (10μl; the concentration
of 1-octanol used was similar to the concentration of 1-octanol present in the natural headspace sample)20 and
yeast hydrolysate solution (YH; 6% w/v, 10μl, a feeding stimulant) were tested individually. e test sample was
pipetted onto lter paper strips that were placed into the treatment cylinder through which air was drawn to one
arm of the olfactometer, while the cylinder through which air was drawn to the control arm of the olfactometer
contained only YH (10μl). Fly activity was video recorded. e time spent in each arm was analysed using
BORIS soware ver. 7.9.647. Twenty replicates were conducted for each type of sample.
Oviposition assay. To determine whether 1-octanol is key in deterring oviposition by gravid female ies,
oviposition responses of gravid females were assessed using agarose plates containing an oviposition stimulant
(OS; γ-octalactone)44. Number of eggs oviposited on agarose plates containing synthetic blends of weaver ant
headspace volatiles (BL+OL, BL−OL; 10µl) or 1-octanol (OL; 1.17% v/v in hexane; 10µl) was compared with
number of eggs oviposited on agarose plates containing OS alone (control). Agarose (0.8g in 100ml water) was
melted in a microwave oven, and then cooled to ~ 60°C. OS (0.05% v/v in hexane; 10µl) alone or in combination
with BL+OL, BL−OL or OL (10µl) was added. is mixture was poured into pre-cooled Petri dishes, covered, and
stored for 10min at 4°C. Agarose plates containing OS alone (control) and OS combined with BL+OL, BL−OL and
OL were all provided to gravid females at the same time as a multiple-choice test (50 gravid females; 13–15days
old from mixed sex cages) in mesh cages (45 × 45 × 45cm, BugDorm-4S4545). e plates were placed at four
corners of the mesh cage and were separated by ~ 40cm from each other. Aer 24h, eggs laid in each plate were
counted under a dissecting microscope (Olympus SZX12, Japan). Ten replicates of the assay were conducted.
Statistical analysis. Data from olfactometer assays were subjected to paired t tests to assess whether the
amount of time spent by ies in the olfactometer arms diered signicantly between control and treatment.
Data from oviposition assays were subjected to one-way ANOVA followed by Tukey’s multiple comparison test
to compare the treatments. Statistical analysis was preformed using GraphPad Prism, version 9.0 (GraphPad
Soware LLC, USA).
Data availability
e datasets generated and analysed during this current study are available from ResearchGate (https:// doi. org/
10. 13140/ RG.2. 2. 20780. 74882).
Received: 6 February 2022; Accepted: 8 September 2022
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Acknowledgements
e authors thank Dr. Stefano De Faveri, Department of Agriculture & Fisheries, Mareeba, Queensland for
providing lab space and equipment for work with green tree ants. is research was supported by funds from
Australian Research Council Industrial Transformation Training Centre for Fruit Fly Biosecurity Innovation
(Project IC150100026) including a PhD scholarship for V.K. and a Research Fellowship for S.J.P. is research
received additional support from the SITplus collaborative fruit y program. Project Raising Q-y Sterile Insect
Technique to World Standard (HG14033) is funded by the Hort Frontiers Fruit Fly Fund, part of the Hort
Frontiers strategic partnership initiative developed by Hort Innovation, with co-investment from Macquarie
University and contributions from the Australian Government.
Author contributions
V.K. and P.W.T designed the study, V.K, S.J.P and D.C conducted bioassays and other experiments. V.K. wrote
the rst dra of the manuscript and all authors contributed to the nal version of the manuscript.
Competing interests
e authors declare no competing interests.
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Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
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