Insects. We collected G. aurantianum larvae on the Buena Vista Farm, located in the Republic of Guatemala (14° 57’ 51’’ N and 91° 59’ 49 W) and kept them in controlled conditions (23 ± 2°C, 75 ± 5% relative humidity and a photoperiod of 12:12 hr (L:D) until the adult stage. The larvae were fed artificial diet (García and Parra, 1999), and after emergence, the adults were fed a 10% sucrose solution. For all the tests, we used 2-5 day-old virgin specimens.
Plant material. Plant material was collected from M. integrifolia cultivar Kau which it is considered highly susceptible to the nut borer. Two sizes of macadamia fruits were collected: small (2 cm diameter) and large (3 cm), to determine whether females have some preference in terms of fruit size. In addition, flowers and leaves were collected in the same site as mentioned above.
Collection of volatiles. Volatiles were collected using the method of dynamic headspace. The samples (fruits, flowers, and leaves) were placed inside of a glass aeration chamber (30 x 60 cm). Volatiles were collected by airflow passage at 0.5 L/min, (previously purified by an activated carbon filter) over the plant material. Volatiles were collected in a glass column contained 200 mg Porapak Q 50–80 (Waters Corporation, Milford, MA). Volatiles were collected for a period of 24 h at a temperature of 26 ± 2°C, RH 70 ± 5%, and a photoperiod of 12:12 hr (L:D). Later, the volatiles were eluted from the column with 400 µL dichloromethane which were concentrated to 100 µL under a nitrogen flow and stored in a 1ml glass vial at -20°C until use.
Bioassays. The response of G. aurantianum adults to volatiles collected from fruits, flowers and leaves were evaluated in a glass Y-tube olfactometer (trunk of the Y-tube 15.52 cm long and the two arms 12 cm long, at 45°, internal diameter 2.5 cm). The plant volatile extracts and the dichloromethane (control) were placed at the ends of each arm (1 µL), and later, humidified air filtered with activated carbon was made to pass through each of the arms, while the insect was introduced at the base of the trunk of the olfactometer. A positive response was recorded when the insect crossed the “line of election” (7 cm after passing the intersection of the olfactometer arms) and remained at the site for 10 seconds. Each bioassay had a duration of 5 min, and those insects that did not select after 5 min were excluded from the analysis. A total of 30 replications were performed per treatment. The bioassays were conducted in a dark room at a temperature of 25 ± 1°C between 18:00 and 22:00 hr. The room was illuminated by a red light at a distance of 120 cm (8-10 Lux). During all the tests, the sample and the control were interspersed to avoid biases in the responses. After five replications, the olfactometer was washed with water, soap and acetone and dried in an oven at 90°C for one hour.
Electroantennography. The antennal response of G. aurantianum females and males to M. integrifolia volatiles, to E8-12: Ac and ocimene was determined by electroantennography (EAG). The head of the insect was carefully cut, and the reference electrode was inserted into its base, using a glass capillary, which was filled with a physiological saline solution. A segment of the antenna was cut, and the distal end of the antenna was inserted at the end of the glass capillary placed inside the recording electrode. The signals generated by the antenna passed through a high impedance amplifier (NL 1200; Syntech, Hilversum, The Netherlands) and visualized in a monitor using Syntech software to process the EAG signals. A regulator of stimulus flows (CS-05; Syntech) was used to generate stimuli at intervals of 1 min. A constant current of pure humidified air (0.7 l/min) was directed over the antenna through a glass tube 10 mm in diameter into which the sample was placed for its analysis. A standard aliquot (1 µl) of each plant extract was loaded in a piece of filter paper (0.5 x 3.0 cm, Whatman, No. 1; Whatman International, Maidstone, United Kingdom) exposed to air for 30 s to allow the solvent to evaporate, then inserted into a glass Pasteur pipette or sample cartridge and left for 40 s before applying. A new cartridge was prepared for each antenna replicate. A cartridge with a piece of filter paper loaded with 1 µl of dichloromethane was used as control. A current of humidified pure air (0.7 l/min) was constantly directed onto the antenna through a 10 mm diameter glass tube. To present a stimulus, the pipette tip containing the test compound was inserted through a side hole located at the midpoint of a glass tube through which humidified pure air flowed at 0.5 l/min. A stimulus flow controller (CS-05; Syntech) was used to generate a stimulus at 1 min intervals. The duration of stimulus was 1 s. The continuous flow of clean air through the airflow tube and over the preparation ensured that odors were removed immediately from the vicinity. The plant extract was presented in random order and the test doses for ocimene were presented sequentially from the lowest to the highest concentration (0.01, 0.1, 1, 10 y 100 µg/µl). Control (dichloromethane) stimuli were presented at the beginning and end of each EAG analysis. One replicate was made with one antenna. Each plant extract and ocimene were tested on 2-5 day-old 10 males and 10 females of G. aurantianum.
Gas chromatography coupled to an electroantenodetector (CG-EAD). To determine the antennal active compounds in the extracts, a gas chromatograph (Shimadzu GC-2010 Plus, Tokyo, Japan) coupled with an electroantenodetector (EAD, Syntech, Hilversum, The Netherlands), equipped with a non-polar Factor Four VF-5ms capillary column 30 m long with 0.25 mm internal diameter and a flame ionization detector (FID), as well as a split/splitless injector was used. The samples were injected in splitless mode, using hydrogen as the carrier gas at a flow of 2.0 ml/min. The effluent of the capillary column was divided into two parts (1:1) using a transfer line (glass capillary with no phase) that was connected to the FID and the other to the EAD. The samples were analyzed using a temperature schedule for the chromatograph oven: initial temperature 50°C for 2 min, increasing 5°C/min up to 250°C, with an isotherm for 10 min. The injector temperature was 200°C and that of the detector was 250°C. The signals generated by the antenna and by the FID detector passed through a high incidence amplifier (NL 1200, Syntech, Hilversum, The Netherlands) and were registered with a monitor using the software Syntech version 2.6 (1993-2003) to process the GC-EAD signals. The antennae, previously cut at their base, were inserted into two glass capillaries (for the base and the end of the antennal flagellum) provided with a saline solution. A total of 10 replications of each sample (fruits, flowers and leaves) were analyzed, using an antenna from a different specimen in each replication. Compounds were considered antennally active if they showed an incidence rate of ≥ 60% in each sample analyzed.
Identification of volatiles. The compounds that were antennally active by GC-EAD were identified in a Varian Star 3400 CX gas chromatograph coupled to a Varian selective mass detector, model Saturn 4D (Palo Alto, CA, USA). The compounds were separated using a column of methyl silicone (DB5-MS) 30 m long by 0.25 interior diameter. The samples were injected in splitless mode. A program of 50°C initial temperature (2 min) to a final temperature of 280°C (2 min) with increments of 15°C per min. Helium was used as the carrier gas. Volatiles were analyzed using Saturn GC/MS Workstation software. Compounds were identified by comparing retention times and mass spectra of the available synthetic standards. Other compounds were identified tentatively based on comparison with the spectra library of the National Institute of Standards and Technology 2.0 (NIST) and retention indexes with those reported in the literature.
Chemicals. (E)-8-dodecenyl acetate (Alfa Chemistry, NY, USA), (E)-β-ocimene (Cayman Chemicals, Michigan, USA), ocimene (mix of isomers) (Aldrich, Toluca, Mexico).
Field tests. This assay was conducted on the Buena Vista Farm, municipality of San Pablo, department of San Marcos, Guatemala. White delta-type sticky traps (12 x 18 cm) were used to capture G. aurantianum. These traps were baited with rubber septa that contained ocimene (10 mg), (E)-8-dodecenyl acetate (1 mg) and a mixture of the two in different proportions (1:1, 5:1 and 10:1 mg/mg); entomological glue was placed on the bottom of the traps. The trapping design was completely random blocks; in each block baited traps were placed, and for each treatment five blocks were installed. The blocks were set out in parallel lines with a separation of 50 cm between blocks. The traps were placed at a height of 3 m above the ground with separation of 50 m between traps. The traps were examined every week, and the moths captured per treatment were collected and recorded. After each examination, the traps were rotated within the blocks, while the baits were renewed every 14 days. Sexing was performed using the taxonomic keys employed by Adamski and Brown (2001).
Statistical analysis. The data obtained in the olfactometry tests in “Y” tubes were analyzed using the G-test with Williams’ correction. The field capture data and the EAG responses were analyzed using a one-way ANOVA. Field capture data was transformed with Log (x + 0.5) to comply with the assumptions of normality and homoscedasticity. The comparison of means was performed using the HSD Tukey test (α = 0.5). In both analyses, the value P < 0.05 was considered statistically significant. Data were analyzed using the program R (v. 4.0.5).