Key message

  • This is the first time a defensive response in sweet pepper plants induced by the phytophagy of the generalist predator, Orius laevigatus, has been described. The results further show how the plant’s response to O. laevigatus increases emission of herbivore-induced plant volatiles (HIPVs) which can modulate the behavior of other arthropods (Bemisia tabaci, Frankliniella occidentalis and Encarsia formosa). The results could explain the great success achieved by IPM programs based on the release, establishment and conservation of O. laevigatus in sweet pepper crops.

Introduction

Many species of the genus Orius Wolff, 1811 are considered to be important beneficial insects for a variety of agroecosystems (Hernández and Stonedahl 1999). Orius species are highly polyphagous, yet show preferential tendencies for immature and adult thrips (Chambers et al. 1993; Riudavets 1995; Frescata and Mexia 1996) and to a lesser extent eggs, nymphs and adult whiteflies (Gerling et al. 2001; Arnó et al. 2008). As a result of these prey preferences, some species of Orius are commercially produced for augmentative release as biological control agents in various crops worldwide (Chambers et al. 1993; Sanchez and Lacasa 2002; van Lenteren and Bueno 2003; van Lenteren et al. 2017). In Europe, the minute pirate bug Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) has become the most successful Orius species for biological control, and it is hence widely used in augmentative release programs. The prey range of O. laevigatus is broad and includes agricultural pests such as aphids, whiteflies, lepidopteran eggs, mites and thrips with a distinct preference for the latter group of pests (Venzon et al. 2002; van Lenteren and Bueno 2003). In southeastern Spain, the release of O. laevigatus together with the predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) is key to successful pest management in more than 10,000 ha of protected sweet pepper crops (van der Blom et al. 1997; Sanchez et al. 2000; Calvo et al. 2009; van der Blom et al. 2009).

The genus Orius can also feed on plants, for example on the pollen and sap, which contributes to population survival in the absence of a primary source of protein-rich food in the crop (Cocuzza et al. 1997; Armer et al. 1998). Indeed, O. laevigatus can complete its development by feeding exclusively on fresh pollen from sweet pepper (Vacante et al. 1997). Armer et al. (1998) demonstrated that Orius species can also feed on xylem and mesophyll contents, which consist of mostly water with small amounts of sugars, starch and amino acids. The presence of amylase in O. insidiosus indicates that it can digest starch and therefore can take advantage of feeding directly on the plant (Zeng and Cohen 2000). Plants are not only a source of nutrients but also a substrate for oviposition. Orius females insert their eggs underneath the cuticle in the epidermal to subepidermal cell layers, which enhances offspring survival (Lundgren et al. 2008). During the early developmental stages, O. laevigatus can take nutrients from phloem tissue and survive solely from plant material for several days (Lundgren et al. 2008). All this supports that feeding on plant material is common and ecologically relevant for omnivorous Orius.

Whereas plant-mediated interactions among herbivores have been relatively well studied, the effect of phytophagy by omnivorous predators on plant defenses and the subsequently induced insect behavior has not been investigated until recently. De Puysseleyr et al. (2011) showed that increased tomato plant resistance to F. occidentalis feeding was produced via the jasmonic acid (JA)-mediated wound response during O. laevigatus ovipositing. More recently, several papers have demonstrated the capacity of different species of zoophytophagous species (plant-feeding carnivore), including mirid bugs (Hemiptera: Miridae), to induce plant responses in tomato plants (Pérez-Hedo et al. 2015a, b; Pappas et al. 2015, 2016).

It is hence hypothesized that O. laevigatus would be able to induce plant responses in sweet pepper as has been demonstrated in other plant–zoophytophage systems. As a first step to better understand the interaction between O. laevigatus and sweet pepper, the behavior of O. laevigatus on the plants was studied and plant feeding behavior quantified to compare general behaviors. A series of experiments were then conducted to determine whether O. laevigatus feeding punctures on sweet pepper induce plant defense responses and whether these in turn lead to behavioral responses in pest and natural enemy species. In parallel, targeted gene expression analysis was used on plants previously exposed to O. laevigatus to ascertain which signaling pathways could be involved in plant defensive responses. Finally, the volatile compounds released as part of the plant response to O. laevigatus feeding punctures were characterized.

Materials and methods

Insects and plants

Adult O. laevigatus, the whitefly B. tabaci (Gennadius) (Hemiptera: Aleyrodidae) and the parasitoid Encarsia formosa (Gahan) (Hymenoptera: Aphelinidae) were supplied by Koppert Biological Systems, S.L. (Águilas, Murcia, Spain), all were less than 5 days old. Frankliniella occidentalis adults were obtained from a colony established at Instituto Valenciano de Investigaciones Agrarias (IVIA) in 2010 and originally collected from Campo de Cartagena (Murcia, Spain). Thrips colonies were maintained on the common bean (Phaseolus vulgaris L. Fabales: Fabaceae) and housed in a climatic chamber at 25 ± 2 °C, 65 ± 10% RH and a 14:10 h (L:D) photoperiod at IVIA.

Pesticide-free sweet pepper seedlings cv (‘Lipari’) [Capsicum annuum (Solanaceae)] (Dulce italiano, Mascarell semillas S.L., Valencia, Spain) were individually transplanted to plastic pots (8 × 8 × 8 cm) containing a mixture of natural soil with local peat moss and housed undisturbed in climatic chamber at 25 ± 2 °C, 60–80% RH and 14:10 h (L:D) photoperiod at IVIA. Once the sweet pepper plants had approximately six fully developed leaves (approximately 15 cm in height), they were used for the study of O. laevigatus behavior, and older plants of approximately 20 cm in height were used for the rest of the experiments.

To obtain O. laevigatus-punctured plants, four intact sweet pepper plants were enclosed for 24 h in a 60 × 60 × 60 cm plastic cage (BugDorm-2 insect tents; MegaView Science Co., Ltd., Taichung, Taiwan) and exposed to 100 O. laevigatus adults, all less than 4 days old (sex ratio 1:1). All individuals were removed from the plants before the experiment. Prior to their use, O. laevigatus were released into a plastic cage (30 × 30 × 30 cm) (BugDorm-1 insect tents; MegaView Science Co., Ltd., Taichung, Taiwan) with water supplied on soaked cotton plugs and starved for 24 h.

Behavior of O. laevigatus on sweet pepper

The behaviors of both male and female O. laevigatus on sweet pepper plants in the absence of prey items were observed during 30-min assays. Less than 4-day-old males and presumably mated females of O. laevigatus were individually placed inside a plastic 5-ml vial and starved for 24 h before use. Water was supplied on soaked cotton plugs.

A clean sweet pepper plant was placed inside a 60 × 60 × 60 cm plastic cage (BugDorm-2 insect tents). A single predatory O. laevigatus (male or female) was gently released and observations began once the individual walked freely onto the plant for the first time. The different observed behaviors were recorded continuously for 30 min. Behavioral observations were made by three researchers using a handheld magnifying glass after first quantifying their reliability. The sweet pepper plant was replaced before the start of each behavioral assay. Assays were repeated until 20 replicates for each sex had been observed.

Locations were defined as off plant (plastic cage, plastic pot or soil) and on plant (distinguishing between apical and basal regions). The apical region was considered the first 5 cm of the plant formed by the apical stem, young leaves and two fully developed leaves. Conversely, the rest of the plant, approximately 10 cm with four fully developed leaves, basal stem and cotyledons, represented the basal region. Seven behavioral states were defined as follows:

  • Grooming (G): The predator’s forelegs are used to clean mouthparts or another part of the body.

  • Feeding (F): The predator inserts its stylet into the plant and stylet movements can be observed.

  • Oviposition (O): The predator presses its whole abdomen onto plant and the ovipositor is inserted into the plant.

  • Resting (R): The predator stands motionless.

  • Antennating (A): The predator is at rest but moves its antennae.

  • Walking (W): The predator walks on different regions of the plant without moving its antennae.

  • Walking–Antennating (WA): The predator walks on different regions of the plant and moves its antenna at the same time.

Because the main interest was in quantifying the feeding behavior compared to other behaviors, only the analysis of the timed behaviorals grouped in the apical and basal regions of the plant is presented.

Frankliniella occidentalis and B. tabaci plant selection mediated by O. laevigatus.

A Y-tube olfactometer experiment was conducted to test the olfactory responses of B. tabaci, F. occidentalis and E. formosa females to sweet pepper plants that were previously punctured by O. laevigatus relative to intact plants. The Y-tube olfactometer (Analytical Research Systems, Gainesville, FL) consisted of a 2.4-cm-diameter Y-shaped glass tube with a 13.5-cm long base and two arms each 5.75 cm long (Pérez-Hedo and Urbaneja 2015). Both side arms were connected via high-density polyethylene (HDPE) tubes to two identical glass jars (5 l volume) each of which connected to an air pump that produced a unidirectional humidified airflow at 150 ml/min (Pérez-Hedo and Urbaneja 2015). Four 60-cm fluorescent tubes (OSRAM, L18 W/765, OSRAM GmbH, Germany) were positioned 40 cm above the horizontally disposed Y-shaped glass tube. The light intensity registered 2,516 lx over the Y-tube and was measured using a ceptometer (LP-80 AccuPAR, Decagon Devices, Inc. Pullman, WA, USA). All Y-tube experiments were conducted under the following environmental conditions, 23 ± 2 °C, 60 ± 10% RH.

A single individual female of B. tabaci, F. occidentalis or E. formosa was introduced into the tube (entry array) and observed until she had walked at least 3 cm up one of the arms or until 15 min had elapsed. A minimum of 33 valid replicates from each species were recorded for each pair of odor sources. Females that did not choose a side arm within 15 min were recorded as ‘no-choice’ and were excluded from data analysis. After recording five responses, the Y-tube was rinsed with soap water and acetone and left to dry for 5 min. The odor sources were subsequently switched between the left and right side arms to minimize any spatial effect on choice. Both types of plants (intact and punctured) were used only once to test the response of 10 females and then were replaced with new plants.

Plant gene expression analysis

The apical region of the sweet pepper plants, as defined above, was subjected to targeted gene expression analysis to detect: (1) ASR (abscisic acid stress ripening protein 1) a marker gene for abscisic acid (ABA), (2) PIN2 (wound-induced proteinase inhibitor II precursor) a marker gene for jasmonic acid (JA) and (3) PR1 (basic PR-1 protein precursor) a marker gene for salicilic acid (SA) signalling pathway. Samples of the sweet pepper apical region were collected from both intact and O. laevigatus-punctured plants. After homogenization in liquid nitrogen, total RNA was extracted using TRizol (Invitrogen, CA, USA) according to the manufacturer’s instructions (Pérez-Hedo et al. 2015a; Naselli et al. 2016). After homogenizing the sample with TRIzol™ reagent, chloroform was added to separate RNA of protein and DNA, and subsequent isopropanol and 1.2 Mm NaCl were added to precipitate the RNA. The RNA pellets resulting from the precipitation were washed twice with 70% ethanol, dried at room temperature and eluted in water. The RNA was quantified and then treated with the Turbo DNA-free DNase kit (Applied Biosystems) to eliminate any traces of genomic DNA, according to the manufacturer’s protocol. cDNA was synthesized by adding to the samples (1 µg/µl), RT buffer, 10 µM Oligo dT and Prime Script™ RT Reagent Kit (perfect real time) (TAKARA Bio, CA, USA). The reaction mixture was then incubated in the thermo-cycler for 15 min at 37 °C and for 5 s at 85 °C. Real-time PCR amplifications were performed with Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific, MA, USA). Capsicum annuum forward and reverse specific primers (0.5 µl) were designed and added to 5 µl of SYBR Green/ROX qPCR MM and 1 µl of cDNA and then brought to 10 µl total volume with Milli-Q sterile water. PCR reactions were run in duplicate according to the manufacturer’s recommendations. Quantitative PCR was carried out using the LightCycler® 480 System (Roche Molecular Systems, Inc., Switzerland), and the protocol consisted in 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 56 °C for 30 s and 72 °C for 30 s. Melting curve analysis was performed at 95 °C for 5 s, 60 °C for 1 min and then a continuous increase of temperature until 95 °C finalizing the process. Data acquisition and calculation were performed with the thermal cycler’s software and then were collected and analyzed in Microsoft Excel. Each qPCR data point is the average of eight independent experiments. EF1 (elongation factor-1) was used as a standard control gene for normalization. The nucleotide sequences of the gene-specific primers are described in Table 1.

Table 1 Forward and reverse sequences of ASR, PIN2, PR1 and the constitutive gene EF1
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Composition of volatile blends

Volatile compounds were collected by means of solid-phase microextraction (SPME) and separated and detected by means of gas chromatography coupled to mass spectrometry (GC/MS). Volatiles were adsorbed in a 65-µm PDMS/DVB SPME fiber (polydimethylsiloxane/divinylbenzene; Supelco, Bellefonte, PA, USA). The adsorbent-coated fiber was mounted on an SPME fiber holder and injected through the first septum of the sample container. Agitation of the atmosphere inside the container was achieved by pumping at 5 ml/min using an injected syringe through the second septum of the sample container. Each sample was performed for 3 h. Four replicates for the intact plant and three replicates for O. laevigatus-punctured plant treatments were conducted. After collection, the fiber was retracted into the needle and the SPME device was removed from the container for GC–MS analysis.

Desorption was performed by means of a CombiPAL autosampler (CTC Analytics) at 250 °C for 1 min in splitless mode in the injection port of a 6890 N gas chromatograph coupled to a 5975B mass spectrometer (Agilent Technologies). To prevent cross-contamination, fibers were cleaned after desorption in an SPME fiber conditioning station (CTC Analytics) at 250 °C for 5 min under helium flow. Chromatography was performed on a DB-5 ms (60 m, 0.25 mm, 1.00 µm) column with helium as carrier gas, at a constant flow of 1.2 ml/min. The GC interface and MS source temperatures were 260 and 230 °C, respectively. Oven programming conditions were 40 °C for 2 min, 5 °C/min ramp until 250 °C and a final hold at 250 °C for 6 min. Data were recorded in the 35–300 m/z range at five scans/s, with electronic impact ionization at 70 eV. Untargeted analysis of the chromatograms was performed with MetAlign software (WUR, http://www.metalign.nl).

Kovats retention indexes (KIs) were calculated for the compounds. Differentially emitted compounds were first tentatively identified by the comparison of their mass spectra with those in the NIST 05 Mass Spectral Library. When available, identity was confirmed by coelution with pure standards (Sigma-Aldrich). To quantify the selected compounds, one specific ion was selected for each compound, and the corresponding peak area from the extracted ion chromatogram was integrated by means of the ChemStation E.02.02 software (Agilent Technologies). The criteria for ion selection were the highest signal-to-noise ratio and specificity to that particular region of the chromatogram to provide good peak integration.

Data analysis

The different behaviors of O. laevigatus on sweet pepper plants and the predator location were analyzed using three-way analysis of variance (ANOVA), taking as factors, sex, observer researcher and either behavior or location. Tukey’s test was used for mean separation at α < 0.05. Since only three of the 20 tested female O. laevigatus oviposited on plants, this behavior was excluded from the analysis. The data from the olfactory responses were analyzed using a χ 2 goodness-of-fit test based on a null model where the odor sources were selected with equal frequency. Individuals who did not make a choice were excluded from the statistical analysis. The data from gene expression analyses and volatile profiling were analyzed using one-tailed Student’s t test (P < 0.05). For volatile profiling, discriminant compounds were determined by the analysis of the data obtained from the untargeted chromatogram and the results expressed as the mean ± standard error.

Results

Behaviors and locations of O. laevigatus on sweet pepper

Orius laevigatus showed preference to the apical region of the sweet pepper plants where the majority of the observed behaviors were performed (F 2,110 = 108.33; P < 0.0001) (Fig. 1). Neither sex nor the observer researcher was found to be significant (F 1,110 = 0.11; P = 0.7415 and F 2,110 = 0.06; P = 0.9374, respectively).

Fig. 1
figure 1

Residence time (mean ± SE) of O. laevigatus adults on sweet pepper plants. Bars with different letters are significantly different (ANOVA, Tukey’s multiple comparison test α < 0.05) (n = 40)

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Orius laevigatus spent 38% of their time feeding on plant tissues, which was significantly higher than the time spent on all other activities (F 5,221 = 21.01; P < 0.0001) (Fig. 2). Neither the sex nor the observer was significant in the statistical analysis of the behaviors (F 1,221 = 0.12; P = 0.724 and F 2,221 = 0.48; P = 0.6211, respectively).

Fig. 2
figure 2

Time (mean ± SE) spent exhibiting different behaviors by O. laevigatus adults on sweet pepper plants during a period of 30 min. Bars with different letters are significantly different (ANOVA, Tukey’s multiple comparison test α < 0.05) (n = 40)

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Frankliniella occidentalis and B. tabaci plant selection mediated by O. laevigatus.

In the Y-tube experiment, females of F. occidentalis showed preference for the odor emitted from intact plants over that from O. laevigatus-punctured plants (χ 2 = 19.931; P < 0.0001) (Fig. 3). In the case of B. tabaci, tested females were also attracted to the odor emitted by intact sweet pepper plants in comparison with O. laevigatus-punctured plants (χ 2 = 17.071; P < 0.0001) (Fig. 3). In contrast to both phytophagous insects, the parasitoid E. formosa significantly chose O. laevigatus-punctured plants over intact plants in the olfactometer assay (χ 2 = 6.250; P = 0.0124) (Fig. 3).

Fig. 3
figure 3

Response in Y-tube olfactometer of females of F. occidentalis (n = 33), B. tabaci (n = 33) and E. formosa (n = 34) to the odor emitted by intact sweet pepper and by sweet pepper plants previously exposed to O. laevigatus. Significant differences based on a χ 2-test are marked using asterisk (P < 0.05)

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Plant gene expression analysis

Transcriptional analysis showed that plant feeding by O. laevigatus on the apical portion of sweet pepper plants increased the expression of the PIN2 gene (JA pathway) (t = 2.161, P = 0.019) and the PR1 gene (SA pathway) (t = 1.835, P = 0.039) (Fig. 4a, b). In contrast, the ASR1 gene (ABA pathway) was not affected in O. laevigatus-punctured plants (t = 0.113, P = 0.455) (Fig. 4c).

Fig. 4
figure 4

Transcriptional response of the defensive genes ASR1 (a), PIN2 (b) and PR1 (c) responsible for the change in level of the phytohormones ABA, JA and SA, respectively, in O. laevigatus-punctured plants. Data are presented as the mean of eight independent analyses of transcript expression relative to a housekeeping gene ±SE (n = 8). Significant differences based on t test are marked with (asterisk) (P < 0.05)

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Composition of volatile blends

Quantitative differences were recorded in the herbivore-induced plant volatiles (HIPVs) profile of O. laevigatus-punctured and intact sweet pepper plants. Untargeted analysis of the volatiles emitted allowed the identification of ten compounds with significantly increased levels in punctured plants, while no compounds with decreased levels were identified. The emission of discriminant compounds increased in the range of twofold to 100-fold and corresponded to terpenoids (1 monoterpenoid, 4 sesquiterpenoids and 1 norisoprenoid), a set of two (Z)-3-hexenyl esters, methyl salicylate and another unknown compound (Table 2).

Table 2 Significant relative levels (fold changes) of the volatiles emitted by O. laevigatus-punctured plants relative to intact plants
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Discussion

Here, for the first time O. laevigatus-induced defensive response in sweet pepper plants due to phytophagous behavior is described. This is of particular interest as the predator O. laevigatus has been one of the most studied and successfully used augmentative biological control agents in sweet pepper (van Lenteren et al. 2017). Furthermore, the modulated behavior of both pest species (B. tabaci and F. occidentalis) and a natural enemy (E. formosa) associated with sweet pepper, in response to an induced plant response by the zoophytophage O. laevigatus, is documented for the first time.

Previous studies on the behavior of several species of Orius under different conditions (availability of prey and/or pollen, quality of the plant as a feeding and/or oviposition substrate, architecture of the plant) have been reported (Fritshe and Tamó 2000; Yano et al. 2005; Jonathan and Fergen 2006; Lundgren et al. 2008). However, to our knowledge, no other study has quantified the time that a specific Orius species spends feeding on sweet pepper. Here, O. laevigatus feeding behaviors are described and quantified and reveal both females and males feed on plant tissue by inserting their stylets, a behavior which represents 38% of time spent on the plant. When comparing this plant feeding behavior with the other observed behaviors under experimental conditions, plant feeding was the preferred activity by O. laevigtus, occupying the greatest proportion of time. Individuals tested in the behavioral experiment were starved 24 h, with access to just water, before their release into the experimental arenas. The reason for both female and male O. laevigatus plant feeding behavior upon their release could well be due to the need to obtain nutrients from the plant and not so much because of the need for hydration. Indeed, the sequential behavior of importance was searching for prey (the sum of the Antennating—A and Walking–Antennating—WA behaviors). The experimental conditions (starved O. laevigatus, the size of sweet pepper plants and the absence of prey after release) were standardized according to conditions O. laevigatus would encounter in the field when released in augmentative biological control programs. The next step would be to quantify the plant feeding behavior in the presence of prey in sweet pepper plants and whether, under this situation, defensive plant responses are also activated.

It is known that predators avoid laying eggs where prey are scarce or absent as in our study (Evans and Dixon 1986; Hemptinne et al. 1992). Nakashima and Hirose (2002) observed that females of O. sauteri (Poppius) oviposited at higher rates in prey-rich patches than in prey-deficient patches. O. laevigatus individuals used in this study were subjected to a very similar procedure before their use (starvation with access to water for 24 h) as in Nakashima and Hirose (2002), and only three of the 20 tested females of O. laevigatus oviposited on plants. The time represented by oviposition occupied just 0.2% of total observation time. These results suggest that plant defense response can be induced by plant feeding behavior in addition to the oviposition behavior described by De Puysseleyer et al. (De Puysseleyr et al. 2011) in tomatoes. Recently, Naselli et al. (2016) demonstrated that all motile stages of the mirid Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) can induce plant responses in tomato plants, although such responses may differ slightly depending on the stage considered. Similarly, knowing whether immature O. laevigatus individuals can trigger defensive responses in sweet peppers would also be of interest.

Most of the plant defense mechanisms against insects are activated by signal transduction pathways mediated by JA, SA and ethylene (Shivaji et al. 2010; Gill et al. 2010). Little had previously been described on the subject of plant responses to the feeding of O. laevigatus before this study. It is shown here that activation of the JA and SA pathways in O. laevigatus-punctured plants is associated with aversion of both arthropod pests, B. tabaci and F. occidentalis, and by contrast, the whitefly parasitoid E. formosa was significantly attracted to O. laevigatus-punctured plants. Pérez-Hedo et al. (2015a) observed that the feeding activity of the predatory mirid N. tenuis on tomato plants activated the ABA and JA signaling pathways, which made plants exposed to mirids less attractive to B. tabaci and more attractive to E. formosa. Additionally, Pérez-Hedo et al. (2015b) showed that three different zoophytophagous predators [N. tenuis, Macrolophus pygmaeus (Rambur) and Dicyphus maroccanus (Wagner) (Hemiptera: Miridae)] had different capacities to induce specific responses in tomato plants. Tomato plants exposed to N. tenuis were less attractive to B. tabaci as mentioned above but also to the lepidopteran Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). By contrast, tomato plants exposed to M. pygmaeus and D. maroccanus were not able to repel B. tabaci and, more interestingly, became more attractive to T. absoluta. All three zoophytophagous mirid predators activated the JA signaling pathways, which resulted in the attraction of the parasitoid E. formosa to punctured tomato plants.

Pappas et al. (2015, 2016) observed that the performance of Tetranychus urticae (Koch) (Acari: Tetranychidae) decreased as a result of the prior exposure of tomato plants to M. pygmaeus. This was accompanied by a locally and systematically increased accumulation of transcripts and activity of proteinase inhibitors that are known to be involved in plant responses. Similarly, a further step in the research with O. laevigatus will be to investigate the performance, by evaluating different life history traits, of B. tabaci and F. occidentalis on O. laevigatus-punctured plants.

It is well documented that non-consumptive effects triggered by arthropod predators can modulate behavior, physiology, development and morphological traits of a subsequently infesting herbivore (Werner and Peacor 2003). This non-consumptive effect relies on the ability of prey to perceive chemicals and visual cues directly emitted by predators, before being preyed. Escape, avoidance, reduced oviposition, reproduction and reduced feeding are some of the solutions prey employ to avoid predation risk (Nomikou et al. 2003; Sendoya et al. 2009; Ninkovic et al. 2013; Wasserberg et al. 2013; Lee et al. 2014). No chemical compound has yet been isolated which could be related to an O. laevigatus chemical cue, since all differentially identified chemical compounds were emitted from the plant. Therefore, the responses of B. tabaci, F. occidentalis and E. formosa were a direct consequence to the odor emitted from the O. laevigatus-punctured sweet pepper plants.

The results presented here showed increased emission of HIPVs in sweet pepper punctured by O. laevigatus compared to intact plants. These volatiles belong to already identified volatile groups activated in plants being injured by true phytophagy, the green leaf volatiles group (GLVs), terpenoids and methyl salicylate (Kessler and Baldwin 2001, 2002; War et al. 2011). It is known that HIPVs are released by most plant species (McCormick et al. 2014). However, each species of plant could emit a specific blend of volatiles and that the relative amounts of HIPVs could vary widely between species and with the type of damage (Kigathi et al. 2009; Ponzio et al. 2014; Ardanuy et al. 2016). In this paper, it has been shown that the particular volatile profile emitted by the O. laevigatus-punctured plant repelled B. tabaci and F. occidentalis and attracted E. formosa.

In summary, these results show that the effectiveness of O. laevigatus as a biological control agent in sweet pepper is due to not just its predatory role but also to its ability to induce plant defensive responses. These results open the doors to new management methods for B. tabaci and F. occidentalis. Once the volatile(s) responsible for the repellency of both pests have been identified, new repellent products can be developed. In parallel, plant breeding programs aimed at obtaining plants with an ability for greater emission of these volatiles could be implemented.

Author contribution

SB, AU and MP-H conceived and designed the research. All authors performed the research, and SB, AU and MP-H wrote the paper. SB, AU, JLR and MP-H analyzed the data. All authors wrote the manuscript.