Cocoons of A. oinophylla and H. rivillei were collected in late March (winter generation) and early July (summer generation) in 2013 from the wine grape plantations of Trentino Region, Italy, and sent to Lund where they were stored at 23 °C, 60% RH and a 17:7 h L:D cycle. Moths from the different generations started to emerge in mid-to-late May and late July, respectively. Newly emerged moths were separated into individual vials to prevent mating and then sexed based on the difference in relative length of antenna. Males have significantly longer antennae than females, as confirmed by dissection of the genitalia (unpublished results). The adults were kept individually until used in experiments.
Reference compounds of different origin and purity were available for the identification work from our laboratory collection of pheromone compounds. For chemical identification and electrophysiological experiments, (Z)-5-dodecenal (Z5–12:Ald), (E)-5-dodecenal (E5–12:Ald), Z5–14:Ald, (E)-5-tetradecenal (E5–14:Ald), Z7–14:Ald and (E)-7-tetradecenal (E7–14:Ald) were prepared by oxidation of corresponding alcohols with pyridinium chlorochromate as described by Corey and Suggs (1975). The corresponding fatty acids and their methyl esters were also prepared from the respective alcohols as described in Wang et al. (2010). For field experiments, Z5–14:Ald (chemical purity 99.6%) and Z7–14:Ald (chemical purity 98.2%) were purchased from Pherobank (Wageningen, The Netherlands), and Z5–12:Ald (chemical purity 98.7%) was prepared in our laboratory from the corresponding alcohol according to Corey and Suggs (1975)
Isolation of Sex Pheromones and Pheromone Precursors
To collect volatiles, an SPME fiber coated with PDMS (7 μm film thickness, Supelco, USA) was inserted into a 5 mL gas tight syringe (Hamilton, Switzerland) to collect the headspace volatiles from 0 to 5 d old virgin female adults contained in the syringe. Volatile samples from the female moths (up to 30 individuals for A. oinophylla, and up to 8 individuals for H. rivillei) were collected repeatedly for 24 h, 48 h or 60 h. Newly emerged females were added into the syringe chamber during the headspace collection. The SPME samples were immediately subjected to GC/MS or GC-EAD analysis.
For the pheromone precursor analysis, 5–6 female abdominal tips dissected from 1- to 2-d old virgin females were combined and extracted with 10 μL chloroform/methanol (2:1 v:v) at room temperature for 24 h. The fatty acyl contents in the lipid extracts were transformed into corresponding methyl esters by base methanolysis as described in Bjostad and Roelofs (1984), and then analyzed by GC/MS.
The electrophysiological activities of the SPME samples and synthetic reference compounds were determined by analysis on an Agilent 7890 gas chromatograph equipped with a flame ionization detector (FID) (Agilent, Santa Clara, California) and an electroantennographic detector (EAD). An HP-INNOWax column (30 m × 0.25 mm i.d., and 0.25 μm film thickness; J&W Scientific, USA) was used in the GC, where the inlet was set at 230 °C, the transfer line was heated at 255 °C and the detector was set at 280 °C. Hydrogen was used as the carrier gas, and at the end of the column a 1:1 division of the GC effluent was directed to the FID and EAD, respectively. After cutting off the tips, the antennae associated with the head of a 1–2 d old male were mounted to a PRG-2 EAG (10x gain) probe (Syntech, Kirchzarten, Germany) using conductive gel (Blågel, Cefar, Malmö, Sweden), and charcoal-filtered and humidified air passed over the antennal preparation. The GC oven was programmed from 80 °C for 1 min, at a rate of 10 °C/min to 210 °C, held for 10 min and then to 230 °C at 10 °C/min for 10 min. In the case of SPME samples, the fiber containing absorbed volatiles was injected into the GC inlet and held for 5 min. Data were collected with the software GC-EAD Pro Version 4.1 (Syntech).
Gas Chromatography/Mass Spectrometry
The SPME samples of volatiles collected from females and the fatty acyl methyl esters from gland extracts were analyzed on an Agilent 5975 mass-selective detector coupled to an Agilent 6890 gas chromatograph (Agilent). An HP-INNOWax column (30 m × 0.25 mm i.d., and 0.25 μm film thickness; J&W Scientific, USA) and a HP-5MS column (30 m × 0.25 mm i.d., and 0.25 μm film thickness; J&W Scientific, USA) were used for the analyses. Helium was used as carrier gas at a constant flow of 0.8 mL/min corresponding to linear velocity of 33 cm/s. The oven temperature program was the same as in the GC-EAD analyses.
For both SPME samples and gland fatty acyl extracts, compounds were identified based on comparison of their retention times and mass spectra with those of synthetic references on both polar and non-polar columns. Double bond positions in the putative fatty acyl pheromone precursors were localized by GC/MS analysis of the dimethyldisulphide (DMDS) adducts of corresponding methyl esters, prepared according to Dunkelblum et al. (1985). For the analysis of DMDS-adducts of fatty acyl precursors the HP-5MS column was used and the oven was programmed at 80 °C for 2 min, then to 140 °C at a rate of 15 °C/min, and finally to 260 °C at 5 °C/min, held for 20 min.
The first field trials were carried out from 29 July to 17 September 2013 for A. oinophylla, and 31 July to 23 September for H. rivillei, in the same vineyards where the cocoons had been collected. The A. oinophylla trapping experiments were carried out in wine grape plantations of the cultivars Chardonnay at Pochi di Salorno, Bolzano, Italy (470 m a.s.l., 46°14’N 11°13E). The field trials on H. rivillei were carried out in a vineyard with the cultivar Pinot gris at Avio, Trento, Italy (124 m a.s.l., 45°41’N 10°55′E). The vineyards received fungicide treatments but insecticides were not applied. Synthetic blends to be tested were prepared in hexane containing 0.02% of the antioxidant butylated hydroxytoluene (BHT) and loaded on the rubber septa (Catalogue no. 224100–020, Wheaton Science Products, Millville, NJ, USA) used as dispensers. For each species, treatments included a ternary mixture (A), three binary mixtures (B-D), three single components (E-G) and hexane alone as solvent control (H), as well as the commercial lures for the olive moth, P. oleae (I) containing Z7–14:Ald (AgriSense LLC) for comparison. The blend ratios as shown in Figs. 4 and 5 were set as found in the fatty acyl precursor composition. Except for the two control groups (H and I), the dosage of treatment A-G was set at 100 μg of the major component per bait, i.e. Z7–14 Ald for A. oinophylla and Z5–12:Ald for H. rivillei.
A second round of field trials was carried out during five weeks (W1-W5) between late May and late June 2014, to compare different dosages of the most active binary mixture for each species.
The actual blend ratios in all the treatments were confirmed by GC-FID before the field test. Five trap replicates were used for each treatment. Delta-traps with sticky inserts (Csalomon, Budapest, Hungary) were baited with the lures and suspended below the vine canopy, with the different treatments in random sequences. Traps within a replicate were randomly placed in two rows of grapevine in the orchard, with a distance of 15 m between traps and 20 m between rows. Traps were inspected once a week and redistributed within each replicate so as to minimize the potential position effect. The trapped moths were identified by their morphological characteristics.
Differences in trap catch between treatments were compared using one-way ANOVA analysis followed by a Least Significant Difference (LSD) test (P < 0.01). All analyses were performed using SPSS ver.16.