Bactrocera tryoni were obtained from a colony that originated from 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 flies were fed yeast hydrolysate, sugar and water ad libitum and were used in experiments when 10 to 15 days old, when sexually mature41. Major workers of O. smaragdina were collected from five different colonies in the vicinity of Mareeba Research Facility, Department of Agriculture and Fisheries, QLD, Australia (17.00724 °S, 145.42984 °E).
Authentic standards of 1-hexanol, decane, p-cymene, D-limonene, γ-terpinene, 1-octanol, dihydromyrcenol, 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 purification.
Collection of body & gland extracts, volatile emissions, and trail extracts
Cuticular compounds, head extracts, gland extracts (Dufour and poison glands), headspace volatiles and trail extracts of O. smaragdina were collected as described by Kempraj et al. (2020)20. For cuticular compounds, individual ants (n = 100) were dipped in 10 mL of hexane for 10 seconds. 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. The extraction time for cuticular compounds and head extract was crucial in achieving differentiation in the compounds extracted. The 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 significant extractions from glands. For gland extracts, Dufour and poison glands were dissected from the abdomen and remnant tissues were carefully removed using fine 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 with an inlet and outlet and were allowed to acclimatize for 30 minutes prior to collection of volatiles. A charcoal filter was connected to the inlet (4 mm ID) of the glass chamber using Tygon tubing (E-3603). The outlet of the glass chamber was connected to a Tenax tube (50 mg, Scientific Instrument Services Inc, Tenax-GR Mesh 60/80, packed in 6 × 50 mm glass tubes) fitted 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 flow rate of 0.5 L/min for 30 minutes 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. The ants were allowed to make a trail on the rinsed section of the mesh for 24h. 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. The 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 filtration 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 concentrated to 0.5 mL. Trail samples were filtered 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.
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 film) fused silica capillary column. Carrier gas was helium (99.999%) at a flow rate of 1 mL/min. An aliquot of 1 µL was injected in splitless mode, with injector temperature set at 270°C. The 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. The ion source and transfer line temperatures were 200°C and 290°C respectively. The 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 identification of compounds, mass fragmentation patterns were compared with NIST library (NIST17-1, NIST17-2, NIST17s) and Kovats retention indices were compared with literature values. The identities of the compounds were confirmed by comparing retention index and fragmentation patterns of each compound with authentic standards.
Coupled Gas Chromatography-Electroantennographic Detection (GC-EAD) recordings were made using Ag-glass microelectrodes filled with electroconductive gel (Spectra 360, Parker Laboratories 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. The base of the head was then fixed to the tip of the gel-filled indifferent electrode. The tip of an antenna was placed in contact with the recording electrode and was slightly inserted into the gel to stabilize the antenna. The signals were passed through a high impedance amplifier (UN-06, Syntech, Hilversum, The Netherlands). Headspace samples were tested by injecting of 1 µl of sample into the GC column. Effluent 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 flame ionization detector (FID), using an HP-5 column (30 m, 0.32 mm ID, 0.25 μm film, Agilent, CA, US). The carrier gas was hydrogen (99.999%) (BOC, North Ryde, NSW, Australia) at a flow rate of 3.0 mL/min. The injector temperature was 270 °C. The oven temperature was maintained at 45 °C for 2 min, and then increased to 250 °C at 10 °C min-1. The outputs from the EAG amplifier and the FID were monitored simultaneously by GcEad software 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. The identities of FID peaks were confirmed 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. The 16 identified headspace compounds20 were used to prepare two synthetic blends. One synthetic 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 solutions of the headspace compounds with a concentration range of 5.0 – 10.0 mg/mL in hexane were prepared in 10 mL volumetric flasks. The stock solutions were run through GC to obtain response factors for the given concentration. The response factor of undecane was used as a reference to adjust the volumes of each compound added to the synthetic blend. The calculated volumes of the compounds were added to a 10 mL volumetric flask. The flask was filled with hexane to the mark and inverted several times to mix the blend well. The synthetic blend was run through GC to confirm 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. The concentration of undecane, the reference compound, was arbitrary each time but in a range of 10.0 to 15.0 μg/mL. The 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).
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 left to air dry. Experiments were conducted in a controlled environment room (25 ± 2 °C, 60 % RH). To provide traction for the walking insects, filter paper (Whatmann No. 1, 12 cm diameter) was placed on the floor of the central area. The room was illuminated from above by uniform lighting from white LED lights. Individual flies (10–15 days old, without access to food over the preceding 24 h, but with access to water) were introduced to the olfactometer through a hole in the floor. Each fly was given 5 min to acclimatize in the olfactometer, after which the experiment was run for 10 min. The olfactometer was rotated 90° after each replicate to eliminate any directional bias. Air was drawn through the central hole at 200 ml min−1 and subsequently exhausted into the room. The central arena of the olfactometer was divided into four discrete odour fields 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) and yeast hydrolysate solution (YH; 6% w/v, 10 μl, a feeding stimulant) were tested individually. The test sample was pipetted onto filter 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. The time spent in each arm was analysed using BORIS software ver. 7.9.642. Twenty replicates were conducted for each type of sample.
To determine whether 1-octanol is key in deterring oviposition by gravid female flies, oviposition responses of gravid females were assessed using agarose plates containing an oviposition stimulant (OS; γ-octalactone)39. 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. This 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). The plates were placed at four corners of the mesh cage and were separated by ~40 cm from each other. After 24h, eggs laid in each plate were counted under a dissecting microscope (Olympus SZX12, Japan). Ten replicates of the assay were conducted.
Data from olfactometer assays were subjected to paired t tests to assess whether the amount of time spent by flies in the olfactometer arms differed significantly 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 Software LLC, USA).
The datasets generated and analysed during this current study are available from ResearchGate (DOI: 10.13140/RG.2.2.20780.74882).