Olive fruits and branches from about thirty-five-year-old olive trees of ‘Koroneiki’ cv. used in the experiments were collected from the experimental organic olive orchard of the Laboratory of Olive and Agroecological Production Systems, Hellenic Mediterranean University in Heraklion, Greece (35°19΄03΄΄N, 25°06΄20΄΄E). Laboratory colonies of B. oleae and P. concolor were established by using adults that emerged from B. oleae infested olive fruits collected from the experimental organic olive orchard. Bactrocera oleae flies were fed with a mixture of sugar, yeast flakes, and milk powder (2:1:1 volume ratio), 50% honey solution and water ad libitum. Olive fruit flies used in the experiments were obtained from the laboratory colony reared on olive fruits under controlled conditions (21 ± 1°C, 50–60% R.H. and natural photoperiod). The parasitic wasp P. concolor was reared on B. oleae L2-L3 instar larvae of the laboratory colony. The parasitoid colony was maintained in the laboratory under controlled conditions (21 ± 1°C, 50–60% R.H. and natural photoperiod) and adult parasitoids were fed on 50% honey solution and water. Female parasitoids were assumed mated and used for the behavioral experiments at six to eight days after emergence.

Olive fruits alone and branches were used for the behavioral bioassays as well as for the headspace collection of volatiles (Figure 1). Only olive fruits with yellowish-green and yellowish epidermis color (maturation index 0–1) (Guzmán et al., 2015), which are preferred for oviposition by the olive fruit fly, were used for the experiments while fruits with purple and black epidermis were discarded. All plant material (fruits and branches) acclimated to laboratory conditions (21 ± 1°C, 50–60% R.H.) for 24 h prior to behavioral experiments and VOC collection. Furthermore, B. oleae male and female adults were used in the behavioral experiments. Figure 1 illustrates the treatments used in each of the experiments.

The response of P. concolor wasps to chemical cues emitted by the plant (olive tree) and by the host (olive fruit fly) was tested in a dynamic air-flow Y-tube glass olfactometer (main arm length 10 cm, side arm length 8 cm, _∅1 cm) (Figure 2A) as previously described by Milonas et al. (2019). Each arm of the olfactometer was connected with Teflon tubing to a 2 L cylindrical plexiglass container holding the odor source. Air was pumped (Dymax 5 air-sampling vacuum pump, Supelco, Charles Austen Pumps Ltd., UK) through an active charcoal filter, humidified by passing it through a conical 2 L glass flask contained tap water and split into the two arms of the olfactometer at a flow rate of 30 mL min⁻¹. The olfactometer was placed in a panel covered with black fabric in order to prevent the wasps from visually spotting the tested material in odor containers as well as to prevent light entering from the side. The bioassays were conducted at 21 ± 1°C and 50–60% R.H. using three 18 W cool fluorescent light bulbs above the olfactometer providing uniform lighting. In this two-choice set-up we offered to the wasps two different odor sources (testing pair) to choose from. Naive female parasitoids were individually released at the downwind end of the main arm of the olfactometer and observed for 5 min until making a choice. The first choice of the wasps was used as parameter to estimate the attraction to different odor source offered. A choice was recorded when the parasitoid crossed at least two-thirds of one of the side arms and stayed there for 15 s. When the wasps did not make a choice within 5 min, it was counted as ‘no response’ and excluded from the statistical analysis (Giunti et al., 2016; Ponzio et al., 2016). The number of wasps that did not make any choice was excluded only for the statistical analysis but included in the calculations to determine the percentage of the total wasps’ response. The wasp was used only once and then discarded. After every trial, the position of the odor sources was switched and the olfactometer was rotated to avoid any position bias. The olfactometer was washed with mild soap, hot water and acetone after every replicate. A total of fifteen wasps were released for each plant-pair (experiments with fruits or branches) or insect-pair (experiments with the B. oleae) combination. Each testing pair (bioassay combination) consisted of fifteen or six replicates (plant pairs or insect pairs). In each testing pair, we tested 225 or 90 wasps.

In the behavioral experiments, we tested the attraction of the wasps to volatiles emitted by the olive tree (fruits and branches) and to cues emitted by its host B. oleae (Table 1). The response of the wasps to (i) clean air, (ii) non-infested fruits, (iii) egg-infested fruits, (iv) larval-infested fruits, and (v) mechanical-damaged fruits was tested in order to determine whether B. oleae infestation (egg- or larval-infested fruits) induce the release of volatiles that could attract the wasps. Moreover, the mechanical-damaged fruits (to mimic the oviposition puncture of the B. oleae) were used to test if artificial damaging of the fruits could induce the emission of volatiles that may attract the wasps. To investigate whether the feeding damage by the olive fruit fly alters the volatile blend of the infested olive branches and whether the induction of the volatiles is restricted to the infested fruits or also to the adjacent leaves (systemic induction of the volatiles) we tested the response of the wasps to: (i) clean air, (ii) non-infested branches with fruits, (iii) non-infested branches without the fruits (fruits removed prior testing), (iv) larval-infested branches with fruits, and (v) larval-infested branches without the fruits (fruits removed prior testing). Wasps’ response was tested also to cues left on fruits by B. oleae ovipositing females: (i) clean air, (ii) fruits with the oviposition marking, and (iii) fruits without the oviposition marking (washed fruits). Additionally, we investigated wasps’ response to cues emitted directly by the olive fruit flies: (i) clean air, (ii) B. oleae virgin females, (iii) B. oleae virgin males, (iv) B. oleae mated females, and (v) B. oleae mated males.

To investigate whether B. oleae infestation changes plant volatile emission in fruits and/or branches, and thus P. concolor wasps’ preference, volatiles from the headspace of olive fruits and branches were collected (Figure 2B). Headspace volatiles were collected by enclosing the fruits and branches with the different treatments in an oven bag made by polyethylene terephthalate (PET), with thickness 12 μm, and dimensions 35 × 43 × 43 cm (SANITAS, Sarantis Group, Greece). A tape was wrapped around the top and bottom end to close the oven bag and each of the treatments were left for 30 min for acclimatization prior to volatile collection. In all cases, plant volatile collection was performed by using dynamic headspace sampling as outlined in Anastasaki et al. (2018). Ambient air was purified through an activated charcoal filter (10 cm length × 1.5 cm i.d.) containing 0.5 g of activated charcoal (Merck, Germany) tapped with glass wool (extra fine, Assistent, Germany) by inserting Teflon tubing through an opening in the bottom part of the bag. Air was sucked out and headspace volatiles were collected in Teflon-made traps for four hours (at 20 ± 1°C, 50–65% RH), at a flow rate of 360 mL min⁻¹ (Dymax 5 air-sampling vacuum pump, Supelco, Charles Austen Pumps Ltd., UK) by inserting Teflon tubing through an opening in the upper part of the bag. The Teflon-made traps (5 cm length × 4 mm i.d.) were filled with 75 mg Porapak Q 80/100 mesh (Supelco, Bellefonte, PA, USA), tapped with a 2 mm glass wool and 3 mm Teflon tubes on each end. Prior volatile collection, the Teflon-made traps were sequentially washed with 1 mL of methanol, diethyl ether, and n-pentane (Fisher Chemicals, Bishop, UK) and blown dry with N₂. After use oven bags were discarded. After the collection, the adsorbent traps were extracted immediately with 500 μL of n-pentane and stored in a freezer (−20°C) in a sealed vial with a conical inserter until chemical analysis. Prior to chemical analysis, the samples were concentrated to 100 μL under a gentle stream of nitrogen. Blank collections of the system (oven bag with no plant material) were performed following elution with 500 μL n-pentane as described above. Background volatiles from these experiments were subtracted from treatment collections.

Headspace samples were analyzed in a gas chromatograph (GC) (Nexis™ GC-2030, Shimadzu, Japan) connected to a single quadrupole mass spectrometer (MS) (GCMS-QP2020 NX Shimadzu, Japan). One μL of the extract was used for the analysis and injected in GC–MS instrument. Separation of the analytes was performed with a MEGA-5 MS capillary column (5% diphenyl / 95% dimethyl polysiloxane) with the following dimensions: 30 m length, 0.25 mm i.d., and 0.25 μm film thickness (Mega, Legnano, Milan, Italy). Spitless mode was set for 1 min. The flow rate of the carrier gas helium was 1 mL min⁻¹. The oven temperature was maintained at 50°C for 5 min, increased at a rate of 3°C min⁻¹ to 170°C and at a rate of 20°C min⁻¹ to the final temperature of 250°C. The mass spectrometer was operated in Electron ionization (EI) mode with ion energy of −70 eV, filament current 50 μA and source temperature 200°C. Data acquisition was performed in full scan (MS) with scanning range 40–300 amu. Compounds were identified by comparing their elution order, the mass spectra with those from mass spectra libraries (Adams, 2007; NIST 17, Wiley7) and literature data (Adams, 2007; NIST – WebBook site). We also used as an additional criterion retention index (RI) of a series of n-alkane (C₈-C₂₀). Identified compounds were confirmed by comparing RIs with those from the literature (Adams, 2007; NIST – WebBook site). Wherever possible, RI, retention time and mass spectra were compared with commercial standards. The total ion chromatogram was processed by LAB solutions software (version 4) based on the retention time and mass spectrum. Emission rates were calculated for compounds that were detected in at least 50% of the samples for each treatment. Six replicates per treatment were used. Chemical analysis was performed in Benaki Phytopathological Institute, Kifissia, Greece.

Wasps’ preference to different olfactory stimuli were analyzed using binary logistic regression [i.e., a generalized linear model (GLM)] with a binomial distribution and a logit link function. We used the wasps’ first choice as the response variable, while the plant or insect treatment and the replicates used as fixed factors. Differences in VOC profile were analyzed using Projection to Latent Structures Discriminant Analysis (PLS-DA) (SIMCA 16.0.1, Umetrics AB, Umeå, Sweden). Volatile compounds measured as peak area divided by the fresh weight of the plant part tested. Data were log-transformed, mean-centered and scaled to unit variance before PLS-DA processing. Additionally, for each model, compounds with a variable importance for the projection (VIP) value higher than one was generated as well. VIP values estimate the importance of each variable (compound) in the projection and is often used for variable selection. Moreover, the composition of the volatile blend was analyzed using a cluster analysis and data visualized with heatmaps (MetaboAnalyst 5.0). Clustering was performed using Ward’s clustering algorithm with Euclidean distances. Data were log-transformed, range scaled and averaged per treatment. To assess the differences regarding volatile emission per compound between non-infested, B. oleae-infested and mechanically damaged fruits and branches, we used one-way ANOVA with Tukey’s post hoc tests for pairwise comparisons or Kruskal-Wallis H tests depending on data normality. Plant treatment used as the independent variable and the volatile emission per compound as the dependent variable. Data normality was assessed by the Shapiro–Wilk test. Statistical analyses were performed using IBM SPSS Statistics 28.0 (IBM corp.).

When testing the wasps’ responses to volatiles from non-infested fruits against clean air, the wasps significantly preferred volatiles of non-infested fruits (Figure 3A; GLM, x² = 90.652, df = 1, p ≤ 0.001). Psyttalia concolor wasps were more attracted to volatiles emitted by B. oleae larval-infested fruits (HIPVs) (Figure 3A) when tested either against clean air (GLM, x² = 77.984, df = 1, p ≤ 0.001), non-infested fruits (GLM, x² = 160.747, df = 1, p ≤ 0.001), or mechanical-damaged fruits (GLM, x² = 129.091, df = 1, p ≤ 0.001). In contrast, volatiles of B. oleae egg-infested fruits (OIPVs) did not influence the attraction of the wasps when tested against volatiles of non-infested fruits (Figure 3A; GLM, x² = 0.786, df = 1, p = 0. 074).

Psyttalia concolor wasps were not attracted to volatiles from the differently treated branches (Figure 3B), neither when testing branches (a) with non-infested fruits (GLM, x² = 0.182, df = 1, p = 0.670), (b) with non-infested fruits from which the fruits were removed prior testing (GLM, x² = 0.392, df = 1, p = 0.531), (c) with larval-infested fruits (GLM, x² = 2.754, df = 1, p = 0.097), nor (d) with larval-infested fruits from which the fruits were removed prior testing (GLM, x² = 0.971, df = 1, p = 0.325) against clean air. Similarly, we observed no differences in wasps’ preference when testing volatiles of non-infested branches with fruits against larval-infested branches with fruits (GLM, x² = 2.697, df = 1, p = 0.101) or non-infested branches with fruits that have been removed prior testing against larval-infested branches with the fruits that have been removed prior testing (GLM, x² = 2.752, df = 1, p = 0.097).

To test the wasps’ response to cues deposited by female flies on the fruit (potential HMPs), we used fruits that were in contact with ovipositing female flies and tested them against fruits that were washed before testing. The parasitoid was not attracted to cues emitted from the fruits with the oviposition marking cue (GLM, x² = 0.725, df = 1, p = 0.394) nor to fruits without the oviposition marking cue (GLM, x² = 1.492, df = 1, p = 0.222) when tested against clean air. Similarly, the wasps were not able to discriminate between fruits with the oviposition marking cue and fruits without the oviposition marking cue (GLM, x² = 0.000, df = 1, p = 1.000) (Figure 4A). Psyttalia concolor wasps significantly preferred the volatiles emitted by virgin female B. oleae (Figure 4B; GLM, x² = 7.571, df = 1, p = 0.006) and mated female B. oleae (GLM, x² = 6.028, df = 1, p = 0.014) when tested against clean air. In contrast, the parasitoid was neither attracted to cues emitted by virgin males (GLM, x² = 2.724, df = 1, p = 0.099) nor mated males (GLM, x² = 1.599, df = 1, p = 0.206) B. oleae when tested against clean air (Figure 4B).

A total of fifty-one volatile organic compounds were detected in the headspace of the olive fruits alone and forty-five compounds from the olive branches. Most of the detected compounds belong to five major classes of plant volatiles released after herbivory: benzenoids and phenylpropanoids, monoterpenoids, homoterpenoids, sesquiterpenoids, and fatty-acid and amino-acid derivatives (Supplementary Tables S1, S2). Overall, qualitative and quantitative differences were observed in the volatile profiles of οlive fruits alone as well as in branches with or without fruits (Figures 5A, 6A, 7; Supplementary Tables S1, S2; Supplementary Figures S1, S2).

Concerning the headspace volatiles from the olive fruits alone, a clustering analysis revealed that each treatment differently affected the volatile profile by altering the emission of specific compounds (Figure 7). In particular, we found that the volatile profiles of non-infested fruits and larval-infested fruits grouped together as well as those of the egg-infested fruits and mechanical-damaged fruits, suggesting that each of the groups have a similar volatile profile (Figure 7). The projection to latent structures discriminant analysis (PLS-DA) resulted in a model with the first two principal components explained 42.7 and 12.5% of the total variance, respectively, (Figure 5A). The first principal component clearly separates the larval-infested fruits from the other three treatments, suggesting that upon herbivore infestation by B. oleae larvae, the olive fruits respond with changes in the volatile emission. In particular, clustering analysis shows that forty-two compounds were emitted in higher amounts upon B. oleae herbivory, either after egg deposition or feeding damage, while twenty-two compounds were produced in higher amounts after mechanically damaging the fruits (Figure 7). From the forty-two compounds that were emitted in higher amounts upon B. oleae infestation, six compounds were detected only in both egg- and larval-infested fruits and thirteen compounds were specific to larval-infested fruits (Supplementary Table S1). Interestingly, from these nineteen compounds the most important ones, with VIP values >1, that contribute to the separation of the volatile profile between the egg- and larval-infested fruits from the other two treatments were: (E)-β-ocimene, (E,E)-α-farnesene, 2-nonanone, and heptanal (Table 2). Furthermore, the loading plot illustrates the contribution of each of the compounds to the differentiation of the volatile blend among treatments based on the VIP values of the compounds (Figure 5B). More specifically, the model identified nineteen compounds with VIP values >1 (Figure 5B; Table 2), suggesting that these compounds contributed the most to the differences observed between treatments. Interestingly, the volatile emission of eleven compounds out of the total nineteen shown in Table 2 was significantly induced in larval-infested fruits compared to the control treatment (Supplementary Table S1). Thus, the compounds that contributed the most to the separation of the VOC profile of the larval-infested fruits from the controls were: n-nonanal, n-octane, heptanal, dimethyl acetophenone, 1-nonanol, (E)-4,8-dimethylnona-1,3,7-triene, 6-methyl-5-hepten-2-one, 2-nonanone, p-ethyl acetophenone, (E)-β-ocimene, and (E,E)-α-farnesene. While the compounds that contributed the most to the separation of the VOC profile of the larval-infested fruits from all the other treatments were: n-nonanal, n-octane, heptanal, 1-nonanol, 2-nonanone, and (E)-β-ocimene (Table 2; Supplementary Table S1).

Regarding the headspace volatiles from olive branches, clustering analysis revealed that the volatile profiles of the branches with the non-infested fruits and the branches with the larval-infested fruits grouped together indicating that they have a similar volatile profile (Supplementary Figure S3). More specifically, the analysis shows that twelve compounds were emitted in higher amounts from branches with larval-infested fruits, and twenty-four compounds were emitted in higher amounts in branches with larval-infested fruits removed prior testing (Supplementary Figure S3). The PLS-DA shows that the first principal component, which explains 56.3% of the total variance, clearly separates the branches with the non-infested fruits and the branches with the larval-infested fruits from the branches with the non-infested fruits removed prior testing and the branches with the larval-infested fruits removed prior testing (Figure 6A). Moreover, PLS-DA shows that the second principal component, which explains 17.3% of the total variance, clearly separates (a) the branches with the non-infested fruits from the branches with the larval-infested fruits and (b) the branches with the non-infested fruits removed prior testing from the branches with the larval-infested fruits removed prior testing (Figure 6A). This could be evidence that the olive tree responds both locally and systemically to herbivore infestation by B. oleae larvae with changes in volatile emission, both in fruits and leaves. The loading plot shows the contribution of each of the compounds to the differentiation of the volatile blend between different treatments (Figure 6B). In detail, the model identified twenty-one compounds with VIP values >1 which suggests that these compounds contributed the most to the differences observed among the treatments (Figure 6B; Table 3). Notably, the volatile emission of two compounds out of the total twenty-one, namely hexadecanol and heptanal, was significantly induced in the branches with larval-infested fruits (Table 3). Another six compounds, different from the two previous, were emitted in higher amounts in branches with larval-infested fruits removed prior testing (Supplementary Table S2).

In the behavioral bioassays, P. concolor wasps exhibit a strong preference toward volatiles of larval-infested fruits (HIPVs) of the olive ‘Koroneiki’ cv. This is in accordance with a study by Giunti et al. (2016) that found that larval infestation by B. oleae induces HIPVs, in three different cultivars of ‘Frantoio’, ‘Leccino’, and ‘Arbequina’, and enhances their attractiveness to P. concolor. From previous studies it is known that artificial wounding and elicitors in insect oral secretion or oviduct secretions are needed to induce plant volatile changes (Meiners and Hilker, 2000; Acevedo et al., 2015). For instance, fatty acid amino acid conjugates, that found to be synthesized in vitro in the larval body of the fruit fly Drosophila melanogaster (Meigen) (Diptera, Drosophilidae) (Yoshinaga et al., 2014), are known to activate receptor-like kinases which then activate specific signaling transduction pathways that trigger the release of HIPVs. Meiners and Hilker (2000) found that a combination of artificial wounding on the leaves surface and oviduct secretion released together with eggs by the elf leaf beetle Xanthogaleruca luteola (Müller) (Coleoptera, Chrysomelidae), induce the release of OIPVs that are attractive to the egg parasitoid Oomyzus gallerucae (Fonscolombe) (Hymenoptera, Eulophidae). Similarly, we observed that P. concolor wasps were more attracted to volatiles from larval-infested fruits but not to mechanical-damaged fruits, which indicates that wounding alone does not elicit the induction of volatiles that could attract P. concolor wasps. Thereby, oral secretion and/or saliva from the B. oleae larvae likely contains an elicitor that induces the emission of HIPVs that serve as a synomone for P. concolor wasps. Moreover, we observed that the wasps were not attracted to the OIPVs from the egg-infested fruits. This suggests that B. oleae egg-infestation could not serve as an early herbivore cue for this parasitoid species. From the larval parasitoids’ point of view, the detection of the presence of the host eggs in fruits might be too early before the actual host stage is present as P. concolor wasps parasitize the 2^nd and 3^rd instar larvae of B. oleae. Furthermore, a previous study found that the egg-larval parasitoid Fopius arisanus (Sonan) (Hymenoptera, Braconidae) was attracted to volatiles from B. oleae egg-infested fruits (Calvitti et al., 2002), indicating that the OIPVs released after B. oleae egg deposition serve as synomones for this parasitoid wasp. This suggests that secretions from the eggs of the olive fruit fly, during oviposition, could have caused the changes in the emission of VOCs to attract F. arisanus wasps. Although OIPVs are well documented from several plant-host systems (Hilker and Fatouros, 2015), no previous knowledge is available from the olive system. Interestingly, chemical analysis of volatiles from egg-infested fruits revealed that, indeed, B. oleae egg infestation induces changes in VOC emission of the olive fruits and this could hint to a cue possibly used by other parasitoids of the olive fruit fly for host location such as the egg-larval parasitoid F. arisanus.

Our olfactory bioassays showed that P. concolor wasps were attracted to host cues emitted by virgin and mated B. oleae females and might use these cues as kairomones. The ability of parasitoids to eavesdrop on host cues to easily detect their host is well documented (Vet and Dicke, 1992; Fatouros et al., 2008; Benelli et al., 2014a). These chemical cues may be derived from the host either as long-range (i.e., host pheromones) or short-range/contact (e.g., from the wing scales, feces, cuticle) host cues (Vet and Dicke, 1992; Fatouros et al., 2008). The two major sex pheromone components of B. oleae are 1,7-Dioxaspiro[5.5]undecane produced by virgin female and young males, and (Z)-9-Tricosene exclusively produced by sexually mature males but not by young males (Benelli et al., 2014b). When testing the attraction of P. concolor wasps to the synthetic compounds 1,7-Dioxaspiro[5.5]undecane and (Z)-9-Tricosene the wasps were more attracted to (Z)-9-tricosene (Benelli et al., 2014b). However, in our bioassay experiments, we found that the wasps preferred the virgin and mated B. oleae females. The female-derived 1,7-Dioxaspiro[5.5]undecane is a compound with high volatility (Benelli et al., 2014a) and therefore it is unlikely to influence the wasps’ behavior as it is not stay long in the environment. Thus, we suggest that other compounds produced by virgin and mated B. oleae females, e.g., methyl dodecanoate, nonanal, or α-pinene (Benelli et al., 2014b; Canale et al., 2015), could play a role on the wasps’ attraction. Furthermore, we cannot exclude the possibility that other type of pheromones could influence the attraction of the wasps. Several Tephritidae, including B. oleae, appear to exhibit an oviposition marking behavior after egg deposition (Benelli et al., 2014b). Bactrocera oleae females have been observed to use their labellum to take out the secretions of the olive pulp from the oviposition puncture and spread it in the fruit surface which functions as oviposition deterrent by conspecifics (Benelli et al., 2014b). It has been reported that HMPs can be exploited by the parasitoids as kairomones to increase their searching efficiency and thus to find a suitable host for their offspring (Vet and Dicke, 1992; Nufio and Papaj, 2001). However, our data did not support this because when we offered fruits with and without cues left by the ovipositing female fly to P. concolor, the wasps were not able to discriminate between the two odor sources. We hypothesize that due to the non-volatility or low volatility of the cues (Nufio and Papaj, 2001), the wasps may be only able to detect them with their contact chemoreceptors. Moreover, cuticular hydrocarbons (CHCs) that are present on B. oleae (Galhoum, 2017; Scolari et al., 2021) could potentially been left as traces on the surface of the fruits. Egg parasitoids of true bugs are known to respond to footprints of their hosts mediated by the absorption of contact kairomone in the epicuticular wax layer of plants walked upon by the host bugs (Conti and Colazza, 2012). It would be interesting to further investigate this in the B. oleae - P. concolor relationship.

In the present study, we found that larval-infested fruits exclusively emit several benzenoids/phenylpropanoids, sesquiterpenoids and green leaf volatiles, which have been reported to be induced upon herbivory and the volatiles emitted serve as attractants for predators and parasitoids (Kessler and Baldwin, 2002; Mumm and Dicke, 2010). Important qualitative and quantitative differences were detected in the volatile blend of egg- and larval-infested fruits compared to non-infested and mechanically damaged fruits of ‘Koroneiki cv. From the fifty-one identified compounds in treatments with olive fruits alone, forty-two compounds were significantly increased after egg- and larval-infestation. Six compounds were exclusively produced in egg- and larval-infested fruits and thirteen compounds were specific to larval-infested fruits. Two previous studies have reported qualitative and quantitative differences in the volatile blend of the B. oleae larval-infested fruits as well. More specifically, they found that specific volatile compounds significantly increased in the larval-infested fruits compared to non-infested fruits (Alagna et al., 2016; Giunti et al., 2016), while, specific volatile compounds exclusively emitted from the larval-infested fruits (Giunti et al., 2016). In the present study, we found volatile differences between the egg- and larval-infested fruits from the controls and mechanical damaged fruits. These differences were influenced the most by (E)-β-ocimene, (E,E)-α-farnesene, 2-nonanone, and heptanal. Compounds that were only found in the volatile blend of larval-infested fruits were: n-nonanal, n-octane, heptanal, 1-nonanol, 2-nonanone, and (E)-β-ocimene. All three cultivars that Giunti et al. (2016) examined had in common two volatile compounds, (E)-β-ocimene and (E,E)-α-farnesene, that had been induced upon larval infestation. Compounds that were also detected in our study. Additionally, it has been reported that P. concolor wasps were attracted to larval-infested fruits (Giunti et al., 2016). These findings suggest that (E)-β-ocimene and (E,E)-α-farnesene might play a key role during P. concolor host location. It seems that the presence of compounds found to be emitted only by B. oleae infested fruits or those that significantly increased might act as attractants to parasitoid P. concolor. Notably, in our study system we found many qualitative changes in VOC emission upon B. oleae infestation apart of changes in ratios of specific compounds. This is quite interesting, as not many systems report qualitative differences in VOC emission upon infestation, rather changes in VOC ratios. For instance, several studies in apple (Benelli et al., 2013), bean (Birkett et al., 2003), tomato (Milonas et al., 2019), and brassicaceous plants (Fatouros et al., 2012) have reported mainly changes in ratios of the volatile emission rather than qualitative differences in volatile compounds upon larval and egg-infestation.

The knowledge obtained from our study could be used in biological pest control programs to improve parasitoid’s important biological control traits such as searching efficiency, host finding, host recognition, host location, host acceptance, and high parasitization rates (Leung et al., 2020). To improve the wasps’ effectiveness, selection of the appropriate wasp genotypes with the desired traits, through selective breeding, is needed. In such breeding programs, knowledge on intraspecific genetic variation of the desired traits of the candidate biocontrol agent is of great importance as it will define its effectiveness as a natural enemy of a specific insect pest (Lommen et al., 2017; Kruitwagen et al., 2018). To enhance the parasitoid’s efficiency, and thus to improve pest control, various approaches must be considered of such as the use of allelochemical-based tactics in order to enhance the efficiency of the biocontrol agent by manipulating their foraging behavior (Peri et al., 2018). This can be achieved, for instance, by using host kairomones (e.g., sex pheromones) and synthetic HIPVs (plant synomones) that are attractive to the targeted parasitoid to enhance the parasitism rates in the field (Peri et al., 2018), or by manipulating the HIPVs emission of the crops by applying HIPV inducers, such as phytohormones, to prime the plants’ defense response to herbivory (Turlings and Erb, 2018) in order to attract natural enemies.