Plant material
Olives from three different cultivars (Ottobratica, Roggianella and Sinopolese) were collected from organic olive grove placed in southern Italy, in Calabria region, near the town of Delianuova (38°1458.0“N; 15°5509.8“E), on the slopes of Aspromonte mountain massif (elevation ca. 600 m). Olives used for behavioural assays and GC-MS analyses were collected in November 2017 from non-irrigated 20 years old olive trees. Fruits were yielded manually (ca. 5 Kg for every cultivar), stored into glass jars and transferred to laboratory conditions within 3 hours.
The olives were firstly separated according to the maturation index (MI), whereby the skin and flesh colours were scored to a 0 to 7 scale42. Olives with MI from 0 to 1 were scored as “Green”, while the fruits with MI from 3 to 4 were labelled as “Black”. Half-ripe olives (MI = 2) and over-matured fruits with pigmented pulp (MI = 5–7) were discharged from further analyses, as fruits presenting elevated oleuropein levels and high lipidic amount are less attractive for B. oleae oviposition, as well as less suitable for larval development43. Among both Green and Black olive bulks, two different infestation degrees were selected: low-infested [i.e. olives with 1–2 B. oleae oviposition punctures on the epicarp, but no exit holes; LI, hereafter] and high-infested fruits [i.e. olives with 3–4 B. oleae oviposition punctures and 1 exit hole; HI, hereafter]. Thus, both low- and high-infested fruits presented an ongoing infestation, but high-infested olives also bore a previous infestation, ended with the pupation of the fully developed larvae outside the fruit. These infestation levels were chosen also considering that the small drupes from oil variety may usually sustain the development of 2 larvae, before rotting and drying out. The remaining olive fruits, which presented different infestation status, were limited in number and thus not considered for further analyses. Similarly, crushed and naturally damaged olives were discarded from GC-MS analyses and behavioural assays.
Due to a particularly high population density during the 2017, uninfested olives were too few to be considered for further analyses. Previous researches demonstrated that olive fruits collected on different months and/or different seasons can produce different volatilome20,21. Therefore, we decided not to sample uninfested olives on the following season. Nevertheless, it has been demonstrated that uninfested olives differ from infested drupes in term of (E)-β-ocimene emissions20,22,23. Although every olive cultivar presents a different volatilome, exclusively the emission of this monoterpene increases in presence of B. oleae infestation crosswise to the olive varieties.
Here, the drupes from every cultivar and maturation index were split in four treatments: (i) high-infested black olives (HI-Black); (ii) high-infested green olives (HI-Green); (iii) low-infested black olives (LI-Black) and (iv) low-infested green olives (LI-Green). Fruits from different treatments were stored separately into sealed clean glass jars (diameter 10 cm, length 20 cm), to avoid volatile contaminations. Before being tested for GC-MS analyses or behavioural assays, olives were stored at laboratory conditions (20 ± 1 °C, 45–55% R.H.) for 12–36 hours. All olives used for GC-MS analyses and for bioassays were subsequently dissected to check the effective presence of B. oleae larvae inside the fruit.
Insect colony
Bactrocera oleae was reared as described by Canale et al.44. Pupae of B. oleae were obtained from field-collected olives arriving at a pressing plant in Delianuova (RC, Italy) in 2017 from October to November. Pupae (ca 500 per cage) were held in BugDorm-6S610® (MegaView Science Education Services Co., Ltd., Taichung, Taiwan) and maintained under controlled conditions at 22 ± 1 °C, 45–55% RH until adult emergence. Adult flies were maintained in the cages to allow mating. Bactrocera oleae adults were fed on a dry diet of hydrolysed yeast and sucrose (1:10 wt:wt), while water was provided ad libitum on a cotton wick.
Olfactometry
Behavioural assays were conducted at 22 ± 1 °C (45–55% R.H.). The bioassays were performed in a Y-tube olfactometer connected with an air delivery system, equipped with an activated charcoal filter, which blown the purified air at 0.3 L min−1 constant flow. The Y-tube system consisted in a horizontal glass unit with a central tube (100 length × 15 mm diam) and two lateral arms (90 length × 15 mm diam) ending with a spherical trap chamber (50 mm diam). The lateral arms of the Y-tube were connected by Teflon tubes to two Drechsel bottles (250 mL), containing the odorous sources. Similarly, the Drechsel bottles were connected to the air delivery system (Sigma Scientific LLC, Micanopy, FL – USA) through Teflon connections. To exclude the presence of visual cues affecting insect orientation from the surroundings, a preliminary trial was performed using no odorant sources (blank vs blank), and no positional effect was recorded. In addition, all the trials were carried out in a white plastic box (1.5 × 1 × 0.7 m) illuminated from above with cold fluorescent tubes (20 W, 250 lux). To avoid for daily variability and positional effect, replicates of every treatment were carried out over several days (4–5 days) and the olfactometer arms were inverted after every test. The olfactometer was cleaned with warm water (35–40 °C) and mild soap and then rinsed with distilled water every 6 replicates (i.e. 6 tested insects).
Bactrocera oleae mated females were tested after 14–21 days from eclosion and were tested in bioassays only once. Adult female flies were gently placed inside the central arm of the Y-tube from a glass vial and observed for 6 min. Flies remaining unresponsive for 5 min were labelled as no-choice. For each responsive female the latent period (i.e. time spent inside the arena before making a choice) and the choice (i.e. the odour source selected) were recorded. The choice was recorded when a responsive fly went inside one of the spherical trap chambers at the end of the Y-tube arms and remained there for at least 30 seconds.
Preferences of B. oleae females toward olive fruits
For the three tested olive varieties (cv. Ottobratica, Roggianella and Sinopolese), the preferences of B. oleae females toward fruits with different maturation degree and B. oleae-infestation status was evaluated. Four treatments were evaluated for each cultivar: LI-Green; LI-Black; HI-Green; HI-Black. To assess if the different levels of B. oleae-infestation, along with the maturation degree of the fruit, could affect the orientation of B. oleae females toward olive drupes, the following odour sources were compared in Y-tube trials:
- (i)
LI-Green vs LI-Black (4 drupes; ca 10 g);
- (ii)
HI-Green vs HI-Black (4 drupes; ca 10 g);
- (iii)
LI-Green vs HI-Green (4 drupes; ca 10 g);
- (iv)
LI-Black vs HI-Black (4 drupes; ca 10 g).
The above listed comparisons were provided for the three tested cultivars separately. The odorous sources consisted in four olive fruits, which were replaced with four new fruits every three replicates. Thirty responsive adult females were tested individually for each comparison.
Effect of B. oleae infestation and fruit maturation on VOC emissions
For the three tested olive varieties (cv. Ottobratica, Roggianella and Sinopolese), the VOC emissions of the four different treatments used in the bioassays were evaluated. For all treatments, 4 olive fruits (ca. 10 g) were inserted into a 30-mL hermetic glass vial and allowed to equilibrate for 30 min. Sampling was accomplished in an air-conditioned room (22 ± 1 °C) to guarantee a stable temperature. Three replicates (i.e. each containing 4 olive fruits) were performed for all treatments. To sample the headspace of the olives a Supelco® (Bellefonte, PA, USA) SPME device coated with polydimethylsiloxane (PDMS, 100 μm) was used. SPME sampling was performed using the same new fiber, preconditioned according to the manufacturer instructions. After the equilibration time, the fiber was exposed to the headspace for 30 min. Once sampling was finished, the fiber was withdrawn into the needle and transferred to the injection port of the GC-MS system. The fiber was desorbed for 5 minutes. All the SPME sampling and desorption conditions were identical for all the samples. Blanks were performed before first SPME extraction and randomly repeated during each series. Quantitative comparisons of relative peaks areas were performed between the same identified chemicals in different samples.
GC-MS analyses were performed with a Thermo Fisher TRACE 1300 gas chromatograph equipped with a DB-5 capillary column (30 m x 0.25 mm; coating thickness = 0.25 μm) and a Thermo Fisher ISQ LT ion trap mass detector (emission current: 10 microamps; count threshold: 1 count; multiplier offset: 0 volts; scan time: 1.00 second; prescan ionization time: 100 microseconds; scan mass range: 30–300 m/z; ionization mode: EI). The following analytical conditions were used: injector and transfer line temperature at 250 and 240 °C, respectively; oven temperature programmed from 60 to 240 °C at 3 °C min−1; carrier gas, helium at 1 mL min−1; splitless injection. Molecule identification was based on comparison of their linear retention indices (LRI) relative to the series of n-hydrocarbons, comparing the retention times (RT) with those of pure chemicals, and on computer matching against commercial (NIST 98 and ADAMS) and homemade library mass spectra built from pure substances, components of known oils and MS literature data45,46,47.
Behavioural responses of B. oleae females toward selected synthetic VOCs
All pure chemicals used for bioassays [n-hexane (≥96%), β-myrcene (≥98%), limonene (≥99%), β-ocimene (≥98%)] were purchased from Sigma Aldrich (Munich, Germany). These volatiles were selected because, according to statistics, their emissions were significantly altered by infestation status in all the tested cultivars.
Two different concentrations of each synthetic HIPV (1 and 10 μg/μL) were dissolved in n-hexane and offered to adult B. oleae females in Y-tube bioassays. VOC concentrations used in the bioassays were chosen according to previous results on the EAG and GC-EAD responses of B. oleae toward pure chemicals and extracts44,48. Electro-antennographical analyses determine the perception of different stimuli directly by the antennal sensilla, providing biological information on insect perceptions and facilitating the identification of a low and a high concentration to be used in the olfactometer. For each trial, 5 μl of VOC-solution was applied to a filter paper (1.5 × 1.5 cm Whatman no. 1) and, after solvent evaporation (ca. 20 s), it was inserted into a Drechsel bottle and connected to an arm of the Y-tube olfactometer. Equally, the other Y-tube arm was connected to a Drechsel bottle containing a similar filter paper treated with 5 μl of pure n-hexane as control. Following trials were performed comparing in Y-tube 5 μl of VOC-solution at 1 μg/μL and 5 μl of the same VOC at 10 μg/μL.The filter papers were renewed every three replicates. A total of thirty adult female flies were tested for every trial.
Data analyses
All the described statistical analyses were achieved using the software JMP 11®. Concerning the bioassays, for every trial, a likelihood chi-square test with Yates correction (with α = 0.05) was used to compare the proportion of flies choosing a given cue. The latent periods were processed by a non-parametric model, the Mann-Whitney U test, assuming no normality of data by Shapiro-Wilk test.
Before statistical analysis of VOC emissions, the area integration report of every identified compound and chemical class was transformed into Log values. The normal distribution of selected data was also checked using Shapiro–Wilk test (P > 0.05). For every tested cultivar, a GLM with two fixed factors (infestation status and maturation) [yj = μ + Ij + Mj + (Ij × Mj) + ej, in which yj is the observation, μ is the overall mean, Ij the fruit infestation status (j = 1–2), Mj the maturation status (j = 1–2), Ij × Mj the interaction infestation × maturation status, and ej the residual error] was performed, to evaluate possible alteration in volatile emissions between the different treatments. False discovery rate (FDR; Benjamini and Hochberg49) was assessed to control the experiment-wide error rate provided by the large number of comparisons.
To investigate the different sources of VOC variability, which can be attributable to genetic differences (i.e. varieties), as well as to common factors (i.e. infestation status and maturation), Principal Component Analysis (PCA) was achieved on transformed values of each VOC. To reveal clusters related to the original variables (variety, maturation and infestation status), two-dimensional score plots were produced. Next, a Multi-Factorial Analysis (MFA) was performed using a principal component procedure and a VARIMAX orthogonal rotation technique. The rotated factors with an eigenvector of at least ± 0.5 were considered to label each factor according to the involved source of variability. Furthermore, scores of common factors were calculated as described by Macciotta et al.50 and analysed using a GLM with three fixed factors (variety, maturation and infestation status). Step-wise discriminant analyses were also completed to find a set of variables (with R2 > 0.1) highly representative of fixed factors (i.e. infestation status and maturation degree). The ratio (Wilks lambda) between the generalized within-category dispersion and the total dispersion was taken into account51.
Source: Ecology - nature.com
