Infested fruits were collected from a mixed fruit orchard at Cukurova University Research and Application Farm located in the southeast Mediterranean region of Turkey. Emerged adults of C. capitata were cultivated under laboratory conditions (25 ± 2 C and 60–70% relative humidity (RH) and 12h photophase). Adults were provided water and a solid diet that consisted of a three-part sugar and one-part yeast diet. Adults were kept in plexiglass cages. Eggs of C.capitata were collected through a fine-meshed on the front wall of their cage into a trough of water. The larvae were reared on a wheat bran diet (wheat bran 65 g, sugar 30 g, yeast 20 g, HCL 37% 4ml, sodium benzoate 1g, and tap water 127 ml). Then, the last larval stage was placed into the containers containing moist perlite.
Apricot fruits were purchased from a supermarket. Fruits were inspected visually to be free from any infestation. Also, they were doused in water with vinegar for 20 min to remove residues from the surface before headspace collection.
Extraction of chemical compounds
For the extraction of compounds in apricot, the automatic HS-SPME/GC-MS technique was used. Headspace volatiles was collected by using (HS-SPME) fiber coated with a 50/30 μm DVB/CAR/PDMS and concentrated volatiles by their sorption characteristics. The flesh of each apricot was cut into cubes (0.3*0.3*0.3), and 3 g of sample were placed into a vial then 40°C held for 15 min. Solid-phase microextraction (SPME) fiber was inserted into the headspace with continuous heating and agitation for 30 min to adsorb volatile substances. Thermal desorption was conducted in the injector glass liner at 250 °C.
Volatile compounds in the samples were analyzed using GC-MS. In the gas chromatographic system, a DB-Wax (60 m x 0.25mm i.dx 0.25 μm) capillary column was used. Helium was used as a carrier gas with a flow rate of 1 mL/min. The split rate is 1:5. The GC oven temperature program was as follows: 40°C maintained for 4 min, increased to 90°C at 3°C/min, increased at 4°C/min to 130°C and held for 4 min, then increased to 240°C at 5°C/min. MS data were was set to scan mode from m/z = 30 to m/z = 600. The ionizing voltage was 70 eV. Volatiles were identified through mass spectral comparison with the Wiley 8 mass spectral databases.
3-hexen-1-ol, acetate, (Z)- (98%), ethyl acetate (98%), butanoic acid, 2-hexenyl ester, (E)- (98%) were purchased from Sigma-Aldrich (Adana, Turkey).
Four-arm olfactometer bioassay
The response toward apricot volatile odors of C. capitata was tested in a four-armed olfactometer. The olfactometer consisted of a central glass area with four arms, each connected to a gas cleaning bottle. Each arm was connected via silicon tubing to a gas cleaning bottle that contained the odor source. Silicon tubes were used to link the activated carbon filter bottle, vacuum pump, flow meter, and gas cleaning bottle containing the water. To prevent the occurrence of visual disturbances, a 20-W light was placed above the olfactometer in a room at 70% R.H., and 25°C ±2. The bioassay studies were conducted using three-day-old adults. Test insects were kept starved for 24 h before the bioassays. A piece of ﬁlter paper containing at 5% concentration volatile samples or the control (fresh air) was placed into each of the gas-washing bottles. For each assay, one group of 10 fresh adults (5 females+5 males) was introduced into the release portion, and they were observed for 10 min using a stopwatch. Adults were assayed separately and replicated four times. Flies entering an arm within this time were considered ‘responders’. Olfactometer was cleaned thoroughly with 70% ethanol and distilled water before use. Also, arms were rotated (90°) to minimize positional eﬀects.
Wind tunnel bioassays
Our study was carried out in a wind tunnel with a 45 cm × 80 cm × 220 cm glass fight section. Charcoal-filtered air was passed through the chamber at 0.20 cm/s−1 with air temperatures of 24 ±1°C and 70% ± 5% RH. To avoid bias caused by light, the wind tunnel was lit from above by LED lights set at 10 lux. Test insects were kept starved for 24 h prior to the bioassay. Volatile samples for odor delivery were prepared at 5% concentration and transferred to a 20-ml polypropylene vial before testing. This vial was placed on the tripod ahead of 15 cm of the fan. For each treatment, we tested the landing rate of 10 separately released C. capitata that were given 10 minutes to respond to the volatile chemicals. This test was repeated four times. If the adult did not take off, we terminate the trial and consider it a non-responder. Each adult was used only once. At the end of each treatment, the wind tunnel was cleaned with 70% alcohol and distilled water.
This study was conducted in the cropping year 2021 in Adana (37°03′ 98,21′′N,35°36′ 05.73′′E), Turkey. The study was carried out in 3 da planted Clementine mandarin orchard.
The chemicals were evaluated separately in the field to test their attractiveness to C. capitata adults. A 20-ml polypropylene vial with a 5-mm-diameter hole punched into the cap served as a slow-release dispenser. Dispensers contained 0.5 g of cotton and were baited with 5 ml using 5% concentrations of the chemicals to be evaluated as single compounds. The vials were placed in Lastfly traps. Traps were placed at 1.5–2 m height in the open shade of the Clementine mandarin canopy between every third row in the Clementine mandarin orchard with a distance of more than four trees apart. There were four replicates of each treatment. Traps were cleared weekly between 15 July and 10 August 2021. The captured adults were sexed. During the field studies, traps were rotated clockwise to eliminate possible bias because of uneven distributions of C. capitata populations. Unbaited traps were used as control.
All statistical tests were performed on IBM SPSS 23. Data were checked for homogeneity of variance (Levene test) and the normal distribution of all data (Shapiro–Wilk test; P < 0.05) before analysis. Data were transformed using log 10 (x + 1) to satisfy normality assumptions prior to analysis of variance (ANOVA). Wind tunnel and olfactometer bioassays were carried out as completely randomized designs with the 4 test dates as replicates. The field study was conducted as a randomized block design with four replications. The behaviors of the adults in the wind tunnel were analyzed using the Chi-square goodness-of-fit references. Multiple comparisons were performed using Chi-squared tests with a Bonferroni correction. For olfactometer and field assays, significant differences in the number of C. capitata were analyzed using the two-way (sex and chemicals as factors) analysis of variance test (two-way ANOVA) followed by Tukey’s multiple comparison test at P < 0.05. Also, to further understand the effect of chemicals, data from females, males, and both were subjected to seperate a one-way ANOVA (chemicals as factors). Significant ANOVAs were followed by TUKEY’s test at P < 0.05 level. All data in this study are shown as mean ± standard error (SE).