Thrips culture and TSWV inoculation
The spinosad-susceptible strain of F. occidentalis (Ivf03) was a field population that was initially gathered from melon plants (Cucumis melo L.) growing in a green-house at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (CAAS), Beijing, in 2003. This population has since been maintained on kidney bean pods in the lab without exposure to pesticides (Zhang et al. 2007, 2015). The formation and upkeep of the spinosad-resistant strain (NIL-R), which (Yuan et al. 2017) documented, was a near-isogenic line of F. occidentalis Ivf03 with a high level of spinosad resistance (its resistance ratio is 36,000).
Plants of Datura stramonium L. were cultivated separately in 1.5-L containers with Metro mix potting soil. The plants were kept in a pest-free, temperature-controlled glasshouse (26 ± 2°C). To prevent the development of viral variants with decreased transmissibility via thrips transmission, TSWV isolate TSWV-YN (Zhang et al. 2013) was kept on D. stramonium plants. The virus was mechanically implanted onto young D. stramonium leaves. Double-antibody sandwich enzyme-linked immunosorbent test (DAS-ELISA), using a kit provided by Adgen Phyto Diagnostics (Neogen Europe (Ayr), Ltd.) and following the manufacturer's instructions, was used to confirm a systemic infection of the inoculated plants about two weeks after inoculation (Zhao et al. 2017). These DAS-ELISA-positive plants were exploited by freshly hatched F. occidentalis nymphs to acquire the virus. Acquisition of TSWV by F. occidentalis.
Adult thrips colonies were placed in glass jars with fresh bean pods, and the adult females were given 24 hours to deposit their eggs. The bean pods containing the eggs were transferred to fresh jars in a light incubator at 26 ± 1°C with 65–70% relative humidity, a photoperiod of 16 h light: 8 h dark, and the adults were delicately removed with a camel hair brush. Fresh D. stramonium leaves infected with TSWV were placed in petri dishes (15 cm in diameter) on moist filter paper for thrips to acquire TSWV, and newly emerged nymphs (within 8 hours of hatching from eggs) were gently brushed onto healthy and infected. To keep the F. occidentalis nymphs from escaping, the petri dishes were sealed with Parafilm M (Pechiney Plastic Packaging, Inc., USA). During the acquisition access period (AAP), thrips nymphs may eat the Datura leaves until they pupated. Individual thrips were sexed when they were pupae. The trials used the adults as soon as they emerged from the pupae.
Collection and analysis of volatiles
Virgin mature males of both strains, with and without TSWV, were gathered and transferred to a clean tube (1.5ml). To encourage patrolling behaviour, a 60-W tungsten filament lamp was used to shine strong light on the thrips. Headspace volatiles were collected over a 100-m SPME fiber assembly made by Supelco in Bellefonte, Pennsylvania, at a temperature of 27 1°C for two hours. To prepare SPME fibers, they were heated at 220°C for one hour. About 30–40 male thrips from each treatment were used to gather volatiles. The experiment was repeated three times.
Gas chromatography-mass spectroscopy (GC-MS) was used to evaluate SPME samples using a Shimadzu GC-2010 plus GC-MS (Shimadzu, China) fitted with a Restek Corp. Rxi-5MS (length 30 m, inner diameter 0.32 mm, film thickness 0.25 m) column. For 10 minutes, SPME samples were desorbed before the fiber assembly was removed. The GC's temperature programmed were 40°C (hold for 4 min), followed by 8°Cmin − 1 to 250°C (hold for 5 min). By contrasting the mass spectra with the NIST 08 spectra, compounds were identified (National Institute of Standards and Technology, Gaithersburg, Maryland). Using the GC-MS solution software, the integrated peak is as for each peak were calculated and, prior to analysis, corrected for the number of thrips.
Olfactometer experiments
In a Y-tube olfactometer, the response of aggregated pheromones to control and adult female thrips infected with the TSWV virus was evaluated. The interior diameter of the transparent glass Y-tube olfactometer was 5 mm, and it featured two arms that were each 60 mm long and 90° angled. Air was humidified, filtered through activated charcoal, split into two air streams, each of which fed into one arm of the olfactometer at a velocity of 60 mm s-1 and via a glass flask. The test and control odour (clean air) sources were two glass flasks, each measuring 50mL. The glass flasks were illuminated from above by four florescent tubes and by one arm of a fiber optic cold light source at a distance of 40 mm (approximately 10 000 lux). Connections were made with Teflon tubes. Olfactometer experiments was carried out at 25 ± 2 ℃.
A tiny aspirator was used to gather forty mixed-age adult thrips, which were then transferred into the treatment flask and given a carbon dioxide anesthesia to make them unconscious. Using a little brush, test thrips were individually applied to the base of the Y-tube. The choice of each thrips for one of the two odour sources was noted when it passed a line that was 20 mm along either arm after being watched for a maximum of 3 min. If the line did not cross after three minutes, "No choice" was recorded. To eliminate the impact of any setup asymmetries that might have been overlooked, odour sources were switched after five thrips tests. This experiment was repeated four to five times for each odor comparison. The apparatus was cleaned before each test by rinsing with hexane.
Statistical analysis
The chi-square test was used to evaluate olfactometer data in order to find significant deviations (P < 0.05) from an anticipated 1: 1 response (Onagbola and Fadamiro 2011). Females who didn't choose were not included in the analysis. In order to find significant differences in the amounts of each component produced by virus-infected versus non-infected male of F. occidentalis, GCMS data (relative peak area) were ana-lysed using Student's t-test (P < 0.05). Analyses were conducted with SPSS 19.0 software (SPSS Statistics 2010; SPSS Inc., Chicago, IL) with the significance level set at P < 0.05.