Key Message

  • Vicia faba plants detect eggs laid by Halyomorpha halys.

  • Egg-experienced plants show rapid direct defence responses against emerging H. halys brood, with impairment of nymphal development.

  • Egg-experienced plants show rapid activation of genes related to plant resistance.

Introduction

The establishment of the invasive brown marmorated stink bug Halyomorpha halys (Stål) in many European and American agricultural ecosystems poses questions about possible consequences to the local biodiversity and high risks of crop losses (Leskey et al. 2012; Smith et al. 2014). When a polyphagous herbivore establishes in a new area, it engages with potential new plant species and different varieties of known hosts with which it has not yet coevolved (Desurmont et al. 2014). Under this scenario, the outcome of novel plant–herbivore interactions is highly unpredictable. In coevolved communities, plants are able to detect the attacker and respond by activating defence strategies devoted to protecting them (Heil 2014). Conversely, when the system is new, different outcomes may arise as follows: (1) the plant cannot specifically detect the invader, (2) it can detect the invader and act with a response resembling what is exhibited against native herbivore species, or 3) the plant can detect the invader with a novel response (Woodard et al. 2012; Desurmont et al. 2014). In this respect, the ability of the plant to recognize the earlier stages of the pest, and to activate proper defences, may confer a certain level of resistance. Whether or not attacked plants successfully defend themselves against the invader may also determine the success of the invasion (Desurmont et al. 2011).

Plant defences might be directly focused towards the herbivore, e.g. directly killing or impairing development of eggs, larvae or adults, or may be indirect, through the emission of chemical volatiles to recruit herbivore natural enemies (reviewed by Peterson et al. 2016). Both direct and indirect defences can be induced as a consequence of egg laying by the pest, thus representing an early alert used by the plant to activate its defences before the infestation advances (reviewed by Vet and Dicke 1992; Hilker and Meiners 2008; Hilker and Fatouros 2016). Additionally, plants are able to respond to insect oviposition and emit chemicals both when this is associated with mechanical damage, e.g. when the eggs are embedded in the plant tissue (Conti et al. 1996; Conti et al. 1997; Chiappini et al. 2012), and when there is no evident physical damage, i.e. the eggs are glued on the plant surface (Colazza et al. 2004; Conti et al. 2008; Conti et al. 2010; Salerno et al. 2013; reviewed by Conti and Colazza 2012, Hilker and Fatouros 2015). Elicitors present in the eggs or in the exocrine secretions covering the eggs probably activate plant responses (reviewed by Hilker and Fatouros 2015).

Remarkably, in addition to promptly responding to eggs laid on the leaf surface, plants can use insect oviposition as a warning signal of future larval herbivory and thus prepare their defences against the insect offspring (reviewed by Hilker and Fatouros 2015). For instance, Spodoptera exigua Hübner larvae feeding on Nicotiana attenuata Torr. ex Watson plants that carried naturally laid eggs exhibited lower weight and higher mortality compared to larvae developing on egg-free plants (Bandoly et al. 2015). Similarly, larvae of two lepidopteran species that developed on Brassica nigra L. plants previously infested by Pieris brassicae L. eggs weighed less when compared to larvae that developed on egg-free plants (Pashalidou et al. 2013).

Concerning the physiology of plant defences, responses against insects are activated by signal transduction pathways principally mediated by jasmonic acid (JA), salicylic acid (SA) and/or ethylene (Dicke and van Poecke 2002; Zheng and Dicke 2008; Reymond and Farmer 1998; War et al. 2012). As a general rule, attacks by chewing herbivores (i.e. caterpillars or leaf beetles) activate a response in the plant, which is mediated by the JA signalling pathway. On the contrary, piercing-sucking herbivores, like aphids, mostly involve a signalling pathway mediated by SA (Erb et al. 2012; Reymond 2013). However, when exposed to multiple herbivore attacks, plants may respond through different pathways, with interference mechanisms that are also insect-density dependent (Kroes et al. 2014). Concerning stink bugs, two studies demonstrated a possible involvement of both JA and SA as signalling molecules (Peiffer and Felton 2014; Giacometti et al. 2016; reviewed by Giacometti and Zavala 2016).

Chen (2008) provided a comprehensive review of the mechanisms involved in direct defences, including the production of gut-proteinase inhibitors by the plants. Phytophagous insects use digestive enzymes to extract amino acids from the food substrate, and plants might defend by producing proteins that inhibit such enzymes, resulting in amino acid deficiencies and thus increased developmental delay, mortality and possibly reduced fecundity (reviewed by Zhu-Salzman and Zeng 2015). The production of proteinase inhibitors (PI) is induced by herbivore feeding (Chen 2008) and can be primed, i.e. pre-activated, by “warning” signals (Conrath et al. 2015). For instance, airborne volatiles emitted by maize plants attacked by Mythimna separata (Walker) larvae inform the neighbouring plants of a future invasion. Thus, the warned plants prepare themselves by pre-activating a set of genes, including a protease trypsin inhibitor of the Bowman–Birk family (Ali et al. 2013).

Here, we were interested in investigating whether or not the host plant, Vicia faba L., recognizes early infestation by the alien pest H. halys, and if it is able to activate physiological processes that may function as resistance. Using olfactometer bioassays, we previously demonstrated that eggs laid by H. halys are recognized by V. faba plants (Martorana et al. 2017; Rondoni et al. 2017a). Following oviposition, through the emission of herbivore-induced volatile compounds, the plant recruits stink bug egg parasitoids such as Anastatus bifasciatus (Geoffroy) and Ooencyrtus telenomicida (Vassiliev) (Rondoni et al. 2017a). Within this paper, the employment of biological and molecular approaches helped us assess whether eggs, naturally laid by the invasive H. halys, are perceived as a warning signal by V. faba plants and activate a mechanism of enhanced direct defences that might affect the developing progeny.

Materials and methods

Insects

The colony of H. halys was established by individuals collected from the provinces of Milano in 2015 and Modena in 2016, and reared in a controlled conditions chamber (25 ± 1 °C, 60 ± 5% relative humidity, 16 h: 8 h light: dark). Individuals were maintained inside clear plastic food containers (30 × 20 × 15 cm) with 5-cm diameter mesh-covered holes to permit proper aeration.

Approximately 30–40 individuals were placed in each container. A paper towel was spread on the bottom of the cage to provide a hygienic support for food and to allow for further oviposition substrate. Eggs of H. halys were collected every day and transferred to new containers to maintain the colony. All insect stages were provided with a diet consisting of tomato fruits, carrot roots, raw peanuts, sunflower seeds and green bean pods. An upside down cotton sealed jar in a Petri dish was used for water supply. The food was replaced every 2 days, and glass jars for water supply were substituted on a weekly basis.

Plants

Seeds of faba bean plants (Vicia faba L. cv Aguadulce Supersimonia, Fabaceae) were individually sown in plastic pots (9 × 9 × 13 cm) filled with a mixture (1:1:1) of sand, agriperlite (Superlite, Gyproc Saint-Gobain, PPC Italia, Italy) and vermiculite (Silver, Gyproc Saint-Gobain, PPC Italia, Italy). Prior to sowing, seeds were inoculated with Rhizobium symbionts and the nodule presence was verified after the end of the experiment. Plants were maintained in a climatic chamber (24 ± 2 °C, 70 ± 5% RH, 12 h: 12 h L: D) and equally irrigated every three days. A soluble mixture (1.4 g/l) of fertilizer (5-15-45, NPK, Plantfol, Valagro, Italia) was added one week after germination. Three- to four-week-old plants, with approximately 7–8 fully expanded leaves, were used for the experiments.

Biological investigations of direct defences

Biological response of H. halys juveniles to differently treated plants was evaluated. To prepare the different plant treatments and control, a potted plant was placed in a 25 cm × 55 cm sleeve cage made from a 4-mm mesh net kept in place using a plastic support. For plant treatments, one H. halys female was maintained in the cage for 24–72 h and allowed to walk, feed and oviposit on the plant. To assure that stink bugs were provided with a mixed diet and to reduce feeding damage on the plants, about 10 sunflower seeds were placed in a 5-cm diam. Petri dish at the plant base. The different plant treatments and the control were as follows:

  1. 1.

    EGG+ A plant with one H. halys female in ovipositional phase that was allowed to feed and naturally lay eggs on the abaxial surface of a leaf; plants were daily checked for oviposition; once eggs were detected, the female and the cage were removed; and only plants with a single egg mass were used for the experiments;

  2. 2.

    EGG− A plant with one H. halys female in ovipositional phase, which fed on it but did not oviposit within 72 h; subsequently the female and the cage were removed;

  3. 3.

    CNT A clean V. faba plant was used as a control; the plant was caged as above for 72 h but without H. halys female.

After cages were removed, in the cases of EGG− and CNT, a fresh egg batch of H. halys was glued to one side of a cardboard (2 cm × 1 cm size), and the clean side was positioned by a wooden stake on the underside of a leaf. This allowed the hatched nymphs to rapidly reach the plant tissues, simulating what happens under EGG+ conditions, but the eggs were not in contact with the plant. Egg masses were checked multiple times per day to properly pinpoint the hatching time. After nymphal emergence (which occurred within 96 h after oviposition), plants were confined using a cylindrical grid cage (diam: 15 cm; height: 55 cm; mesh size: 1 mm) to avoid the dispersal of the nymphs. Seven days after hatching (T1), six nymphs were randomly collected from each plant, then collectively weighed with a microbalance (Mettler AC 100, Mettler Instrumente AG, Zurich), recorded and removed from the plant. Ten days later (T2), the analyses were repeated on six different nymphs (except for two cases in which five nymphs were available and one case in which only three nymphs were present). After measuring their body weight, all nymphs were frozen and stored at − 20 °C. The length of the hind-tibia of frozen specimens collected at T1 and T2 was then measured by using ImageJ (Rasband 1997). In details, at T1, only second instars were present, whereas at T2, the third instar was the most abundant stage. Therefore, at T2 (17 days after hatching), only the third instar hind-tibia was recorded, since any plant defence that might have reduced the development of the nymphs after T1 should be revealed in those that moulted from second to third instars. Due to the natural mortality of nymphs across all treatments during the experiment, a lower number of replicates were available at T2. Therefore, 12–20 plant replicates were performed at T1 and 6–12 replicates at T2. Plants were maintained in a controlled climatic chamber (25 ± 1 °C, 60 ± 5% relative humidity, 16 h: 8 h light: dark). Data were analysed by means of linear models (at T1 and at T2), testing the hypotheses that the weight and/or the length of the hind-tibia of nymphs that developed on EGG + plants is lower compared to those that developed on EGG− or CNT plants (Zuur et al. 2009).

Molecular investigations of direct defences

To assess the response of differently treated plants after feeding by H. halys juveniles, treatments (EGG+ and EGG−) and control (CNT) were prepared as already mentioned (see above “Biological investigations of direct defences”). Afterwards, plants were kept unchallenged in a controlled conditions chamber for 4 days (96 h), in preparation for the subsequent treatment. During this period, the eggs in the EGG+ treatment were repeatedly checked for hatching. Newly emerged nymphs were carefully removed to prevent their contact with the abaxial leaf tissues.

Each plant was exposed to two groups of mixed instars of H. halys nymphs. Both groups consisted of four nymphs, one each of the second, third, fourth and fifth instars. High infestation load was required in order to assure a possible plant response to the attacks (as reported by Digilio et al. 2010) and was achieved by installing on plants two clip cages containing four nymphs each. A clip cage consisted of a modified Petri dish (5 cm diameter, 1 cm height), with the rim covered by a sponge ring and the bottom ventilated through a mesh-covered hole (as in Rondoni et al. 2017b). The clip was supported by a hairpin attached to a wooden stake inserted into the soil. In the case of the EGG+ treatment, one clip cage was placed on the leaf that carried the egg batch, and the second on the one directly above. For the EGG– treatment and the control, cages were clipped on the third and fourth expanded leaves. In order to describe a trend in gene expression over time, plant exposition to nymphs was maintained for 4, 24, 48 and 72 h. After each respective timeline, the cage and the nymphs were removed and the leaves on which the bugs were held were excised with forceps and frozen at − 80 °C until RNA extraction. Three biological replicates were conducted for each treatment and control, each corresponding to one leaf per plant.

RNA extraction, cDNA synthesis and real-time quantitative RT-PCR

Total RNA from V. faba leaves was extracted using PureLink™ RNA Mini Kit (Invitrogen, USA) as described by the manufacturer. DNase treatment was applied using TURBO™ DNase (Invitrogen, USA) to remove genomic DNA. Treatment success was checked by gel electrophoresis. The RNA concentration was determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). An amount of 500 ng total RNA was used to synthesize the cDNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., USA) according to the manufacturer’s instructions. Gene expression was conducted using SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories Inc., USA) in 20 μl reaction.

The expression of the genes CPI (cysteine proteinase inhibitor) and TPI (trypsin proteinase inhibitor) was investigated, as it was previously found that these genes might be activated following stink bug feeding (Giacometti et al. 2016) and are possibly involved in priming by herbivore feeding (Ali et al. 2013; Bandoly et al. 2015). In addition, the published literature revealed that stink bug feeding may involve JA and SA (Peiffer and Felton 2014; Giacometti et al. 2016). Therefore, two genes, respectively, were chosen as markers for these pathways, NAI1 transcription factor and PR1 (pathogenesis-related gene 1). NAI1 transcription factor regulates ER body formation after pathogen or herbivore attack in a JA-dependent manner (Matsushima et al. 2004; Ocaña et al. 2015). Expression of PR1 is SA-responsive and promotes the formation of PR protein 1. CYP2 (cyclophilin) was used as reference gene (Gutierrez et al. 2011).

Primer sequences (listed in the supplementary information, Table S1) were obtained from the published literature (NAI1: Ocaña et al. 2015; PR1: Cheng et al. 2012; CYP2: Gutierrez et al. 2011) or designed from V. faba transcriptome (CPI and TPI) deposited in Cool Season Food Legume Crop Database Resources (Humann et al. 2016; transcriptome from Ocaña et al. 2015) using Primer3 v. 0.4.0 (http://frodo.wi.mit.edu/primer3/). Primers were used in 400 nM reaction concentration each. Amplification was performed for 40 cycles at an annealing temperature of 62 °C in the CFX-96 real-time PCR detection system (Bio-Rad Laboratories Inc., USA).

Threshold cycles were used to quantify the relative gene expression following the ΔΔCq method (Schmittgen and Livak 2008) after inter-run calibration (as indicated in Hellemans and Vandesompele 2011). Normalized expression was log + 1 transformed and standardized following the procedure in Willems et al. (2008). The Kolmogorov–Smirnov test was then used to verify the normality of log-transformed data. To deal with heterogeneity in the variances, generalized least squares models were used, by assuming a different variance for each time period (4, 24, 48 and 72 h) (Zuur et al. 2009; Rondoni et al. 2012; Staines et al. 2016). Post hoc comparisons were performed, testing the hypotheses that gene expression changes during nymphal feeding within the different treatments (see Goni et al. 2009). P values were corrected using the Benjamini–Hochberg method (Benjamini and Hochberg 1995). Pearson correlations were also calculated between different gene expressions. All analyses were run under R statistical environment (R Core Team 2014), package nlme (Pinheiro et al. 2012).

Results

Biological investigations of direct defences

At T1 (7 days after hatching), H. halys nymphs that developed on EGG+ plants exhibited a lower weight compared to those that developed on EGG− plants (~ 12% reduction, t = 2.52, df = 1, P = 0.008) or CNT plants (~ 10% reduction, t = 1.82, df = 1, P = 0.038) (Fig. 1).

Fig. 1
figure 1

Mean body weight (± SE) of Halyomorpha halys nymphs measured at 7 d (T1) or 17 d after hatching (T2) on EGG+ (oviposition + feeding by H. halys females), EGG– (feeding by H. halys females) and CNT (untreated control) Vicia faba plants. At T1, the mean body weight of nymphs developing on EGG+ plants was significantly lower than those developing on EGG– or CNT plants (P < 0.05). At T2, body weight of nymphs from EGG+ was lower only in comparison with nymphs from EGG– plants (P < 0.05). See text for detailed results

Full size image

At T2 (17 days after hatching), the nymphs that developed on EGG+ plants weighed less when compared with those that developed on EGG− plants (~ 23% reduction, t = 1.91, df = 1, P = 0.034) but not when compared with those that developed on CNT plants (~ 9% reduction, t = 0.74, df = 1, P = 0.23) (Fig. 1). Concerning insect sizes, at T1, the length of the second instar hind-tibia did not differ significantly in EGG+ plants (1.49 ± 0.01 mm) compared to EGG− (1.47 ± 0.01 mm) (t = 1.39, df = 1, P = 0.91) or to CNT plants (1.49 ± 0.01 mm) (t = − 0.35, df = 1, P = 0.36). However, at T2, the hind-tibia of third instars was shorter on EGG+ (2.25 ± 0.02 mm) compared to CNT plants (2.30 ± 0.02 mm) (t = − 1.90, df = 1, P = 0.030), whereas no differences were detected in EGG+ compared to EGG– (2.29 ± 0.02 mm) plants (t = − 1.45, df = 1, P = 0.075).

Molecular investigations of direct defences

CPI gene expression increased in EGG+ plants at 24 h (Z = 4.30, df = 1, P < 0.001), 48 h (Z = 3.24, df = 1, P = 0.001) and 72 h (Z = 3.39, df = 1, P = 0.001) after exposure to feeding by H. halys nymphs, whereas its expression in EGG− and CNT plants remained unchanged over the course of the experiment (P > 0.05 for all comparisons) (Fig. 2). In contrast, TPI expression over time did not exhibit any significant trend neither in the treatments nor in the control plants (P > 0.05 for all comparisons) (Fig. 2).

Fig. 2
figure 2

Relative transcript levels (mean ± SE) of genes for cysteine proteinase inhibitor (CPI), trypsin proteinase inhibitor (TPI), NAI1 transcription factor (NAI1) and pathogenesis-related 1 (PR1) in leaves of EGG+ (oviposition + feeding by H. halys females), EGG– (feeding by H. halys females) and CNT (untreated control) Vicia faba plants after exposure to H. halys nymphs for 4, 24, 48 and 72 h. Significant increase in expression of CPI gene was detected from EGG+ plants after 24, 48 and 72 h (P < 0.001). Significant increase in expression of NAI1 gene was detected from EGG+ plants after 24 and 48 h (P < 0.05). Significant increase in PR1 gene was detected from EGG+ plants after 72 h (P < 0.001) and from CNT plants after 48 h (P < 0.05). See text for detailed results

Full size image

Expression of JA-responsive NAI1 gene in EGG+ plants significantly increased after 24 h (Z = 2.46, df = 1, P = 0.029) and 48 h (Z = 2.43, df = 1, P = 0.029), but not at 72 h (Z = 1.23, df = 1, P = 0.22). Conversely, the expression of NAI1 in EGG− or in CNT plants remained unaltered throughout all exposure times (P > 0.05 for all the comparisons). A significant correlation was found between CPI and NAI1 expression levels in EGG+ plants (r = 0.67, P < 0.001) (Fig. 2).

The expression of the SA-responsive PR1 gene in EGG+ plants was higher only at 72 h (Z = 7.84, df = 1, P < 0.001). No changes were detected in EGG− plants (P > 0.05 for all comparisons). Higher expression of PR1 was detected in CNT plants after 48 h feeding by nymphs (Z = 2.53, df = 1, P = 0.035) (Fig. 2).

Discussion

Our results show that V. faba plants respond to oviposition associated with feeding by H. halys (EGG+), activating physiological processes that eventually result in a defensive response directed against juveniles of the next generation. After 7 days (T1), H. halys nymphs (first and second instars) reared on oviposition-experienced plants (EGG+) had gained less weight compared to plants only subjected to H. halys feeding (EGG−) and to control plants (CNT). After 17 days (T2), the weight of nymphs developing on EGG+ was still lower when compared to EGG− plants but not when compared to control plants, suggesting that prolonged nymph activity in the latter may have induced some defence mechanisms, although delayed and possibly different from the defences activated in EGG+ plants. Remarkably, at T2, the third instars that had developed on EGG+ plants had reached a smaller size (shorter hind-tibiae) compared to nymphs that had developed on control plants.

Overall, these results suggest that an early “warning” mechanism is activated in plants after detection of the herbivore egg mass, thus allowing a swifter defence response against future juveniles feeding on the plants. After a longer exposure to insect feeding, the effect of oviposition-experienced plants on nymph weight becomes less pronounced compared to control plants, possibly because the latter have in turn activated different defence responses, although residual effects are still visible in insect size. Our results are in accordance with previous studies handling similar responses to oviposition in different plant–herbivore systems (Geiselhardt et al. 2013; Beyaert et al. 2012; Pashalidou et al. 2013; Bandoly et al. 2015; Austel et al. 2016) and at least in some cases, these could be interpreted as a priming of direct plant defences. As a consequence of priming, plants respond faster and/or stronger when subsequently challenged by biotic or abiotic stress factors (Conrath 2011).

Our biological investigations and tentative explanations of direct defences at the insect level were supported by molecular investigations at the plant level. Through expression analysis of selected genes, we detected in EGG+ plants an early (between 4 and 24 h) increase in the induction of CPI and NAI1 genes after the offspring started to feed, whereas no changes were observed in EGG– and in control plants. This further suggests that V. faba plants can detect the presence of the eggs and readily activate corresponding genes, allowing a rapid defence response against the future generation emerging from the eggs. The lower weight exhibited by nymphs that fed on EGG+ plants might be consequently due to the early activation of the CPI gene, which might regulate proteins that interfere with the stink bug digestive processes.

The positive correlation between the expression of NAI1 and CPI we detected in our samples supports JA-dependence of both genes and is in agreement with a previous study, where it was demonstrated that airborne JA induces the expression of proteinase inhibitors that contribute to resistance to herbivory (Farmer and Ryan 1990). JA-dependent genes have been previously shown to be the target of early activation by herbivore feeding. Büchel et al. (2012) created an elm EST database to detect egg-induced defence genes and found that genes involved in phytohormone signalling were expressed, indicative of jasmonic acid biosynthesis and activation of jasmonic acid-responsive genes. Tomato leaves that experienced oviposition by Helicoverpa zea exhibited an early activation of JA-responsive gene PIN2 soon after larval feeding (Kim et al. 2012). Peiffer and Felton (2014) punctured the leaf of tomato plants and injected saliva from H. halys, thus eliciting after 24 h a significant upregulation of PIN2 JA-dependent gene but not of PR1 gene.

In our experiment, feeding by nymphs on EGG+ V. faba plants significantly increased the expression of SA-dependent PR1 at 72 h, corresponding to a decrease in the JA-dependent NAI1. Moreover, a significant increase in PR1 was detected also in CNT plants, but at 48 h, suggesting that plants can detect feeding by nymphs without having been experienced by ovipositing females. This supports the general hypothesis that important plant defences induced by stink bug feeding are often mediated via a SA-dependent pathway. Timbó et al. (2014) analysed the proteins that were regulated after feeding by the subtropical brown stink bug, Euschistus heros (F.), in susceptible and resistant soybean varieties and concluded that resistant variety defences involved the shikimic acid pathway, which is SA-dependent. Giacometti et al. (2016) demonstrated that feeding by the southern green stink bug, Nezara viridula L., activates in soybean seeds the synthesis of an early peak of JA accumulation and ethylene emission, followed by upregulation of SA pathway. They concluded that upregulation of SA-dependent genes was responsible for reduced stink bug preference towards attacked plants.

Our data would evidently support the hypothesis of initial priming of JA-dependent CPI and NAI1 genes as a consequence of oviposition and its following induction as a consequence of nymphal attack. However, the interpretation of PR1 expression appears to be more complex. An increase in SA-dependent PR1 follows only at 72 h, after a decrease of JA-dependent genes, possibly as a consequence of negative crosstalk between SA and JA pathways (Reymond and Farmer 1998). Indeed, a previous investigation detected that SA concentration and PR1 expression are negatively correlated with JA-dependent genes (Reymond and Farmer 1998; but see Giacometti et al. 2016 and Leon-Reyes et al. 2009 for the role of high ethylene in the elimination of the negative SA–JA crosstalk). This explanation would be consistent with the increase in PR1 expression in control plants after 24 h of nymph feeding, as no expression of JA-dependent genes occurs in this case. On the other hand, no increase in PR1 expression has been detected for EGG− after feeding by nymphs. This might be explained by a possible earlier activation of SA pathway, and resultant expression of this gene, as a consequence of intense feeding by the female, which did not oviposit and therefore may have spent longer time feeding.

Intriguingly, no increase in expression was found for the TPI gene. Maize plants exposed to airborne volatiles, emitted by neighbouring plants attacked by lepidopteran larvae, primed a Bowman–Birk trypsin inhibitor gene (Ali et al. 2013). Since herbivorous stink bugs rely on gut cysteine proteinases for their digestion and not on trypsin proteinases (reviewed by Terra and Ferreira 1994), we suspect the existence of a plant defence mechanism specifically directed towards H. halys or to stink bugs in general. Our results are in agreement with Giacometti et al. (2016), who revealed an upregulation of cysteine proteinase inhibitor but not Kunitz-type trypsin inhibitor in soybean plants attacked by N. viridula stink bugs.

In this paper, combining biological and molecular investigations, we demonstrated that eggs naturally laid by the invasive H. halys are perceived by V. faba plants, allowing the pre-activation of JA-dependent genes. Additional investigations and specifically designed experiments, however, are necessary to verify whether this mechanism is directly responsible for the reduced development of nymphal offspring. On the other hand, previous studies demonstrated that oviposition by H. halys induces the activation of indirect defences through the emission of volatile compounds which attract native egg parasitoids of the stink bug (Rondoni et al. 2017a). Field studies revealed that stink bug egg parasitoids are attracted by volatiles produced by plants in a JA-dependent pathway (Moraes et al. 2005; Moraes et al. 2008). Early activation of the JA pathway by stink bug oviposition, therefore, may represent an efficient adaptation of plant defence mechanism to prevent damage from feeding nymphs through the combined expression of anti-digestive compounds and attraction of natural enemies. Additional investigation on the underlying mechanisms involved would be important for the evaluation of possible implementation techniques in pest control through the enhancement of plant resistance.

Author contribution statement

GR and EC conceived and designed the research. GR, VB and RM conducted the experiments. GR analysed the data. GR and EC wrote the manuscript. All authors reviewed the paper.