Arthropod-Plant Interactions (2018) 12:123–131 DOI 10.1007/s11829-017-9560-2

ORIGINAL PAPER

Terpenoids dominate the bouquet of volatile organic compounds produced by Passiflora edulis in response to herbivory by Heliconius erato phyllis (: Nymphalidae)

Eliane de O. Borges1,2 · Camila B. C. Martins1 · Rodolfo R. da Silva1 · Paulo H. G. Zarbin1,2

Received: 16 January 2017 / Accepted: 19 August 2017 / Published online: 4 September 2017 © Springer Science+Business Media B.V. 2017

Abstract In response to injury, plants produce volatile plants displayed a peak of emission of (E)-β-ocimene after organic compounds (VOCs) that usually differ depending 72 h, which distinguished them from HB plants. MD plants on the type of damage they have suffered (e.g., mechanical showed a general increase of VOCs versus undamaged damage, herbivory, and oviposition). The objectives of this control plants. Furthermore, it has been suggested that (E)- study were to identify and compare the bouquet of volatiles β-ocimene may be sequestered by larvae of H. erato phyllis emitted by passion vine plants (Passiflora edulis) after as a component of the odoriferous bouquet of the abdom- injury caused by mechanical damage (MD), herbivory inal scent glands present in adult males, which play a role (HB), and oviposition (OV) by the lepidopteran, Heliconius in sexual communication. erato phyllis. Following injury, extracts of plant emissions were collected from each treatment every 24 h for three Keywords Butterfly · (E)-β-ocimene · Homoterpenes · days and were analyzed by GC and GC/MS. Results show Passion vine · Plant– interaction · Terpenes · TMTT that plants emitted 12 volatiles before and after damage, namely terpenoids, ketones, and aldehydes. Although no significant differences were detected between the three Introduction treatments individually, if the entire bouquet of volatiles is analyzed, samples collected at 24 h were different from Volatile organic compounds (VOCs) produced by plants in samples collected at 48 and 72 h. However, terpenoid response to herbivory may attract the enemies of herbivore emission increased significantly in HB plants after 24 h. (Arimura et al. 2009; Pinto-Zevallos et al. 2013) HB plants emitted approximately 6300, 50, 46, 11, 6, and and may activate responses in conspecific plants and other 3.6 times more (3E,7E)-4,8,12-trimethyl-1,3,7,11-tride- organisms, for example, attracting or repelling lepidopteran catetraene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), females for oviposition (De Moraes et al. 2001; Zakir et al. (E)-β-ocimene, (Z)-β-farnesene, (E)-β-caryophyllene, and 2013). In plants, oviposition by herbivorous insects pro- farnesane, respectively, compared to control plants. OV motes the emission of volatile signals, the formation of necrotic tissues, and the production of ovicidal substances (Hilker and Meiners 2006). In the literature, various studies investigate the production of VOCs by plants following Handling Editor: Anna-Karin Borg-Karlson. herbivory and/or oviposition by lepidopterans (Pare´ and & Paulo H. G. Zarbin Tumlinson 1999; Fatouros et al. 2012; Zakir et al. 2013; [email protected] Horas et al. 2014; Hatano et al. 2015). Many plants are able

1 to discriminate between the damage caused by herbivorous Laborato´rio de Semioquı´micos, Departamento de Quı´mica, insects or mechanical damage by recognizing chemical Universidade Federal do Parana´ (UFPR), CP 19081, Curitiba, PR 81531–990, Brazil elicitors present in the oral secretions of herbivores, and by assessing the quantity and quality of tissue damaged, along 2 Departamento de Zoologia, Programa de Po´s-graduac¸a˜oem Entomologia, Universidade Federal do Parana´ (UFPR), with the pattern, frequency, and the period of feeding by CP 19020, Curitiba, PR 81531-980, Brazil herbivores (Wu and Baldwin 2009). 123 124 E. de O. Borges et al.

Passiflora edulis Sims, 1818 (Malpighiales, Passiflo- their production methodology and the lineages involved are raceae) produces the yellow passion fruit and is the host unknown. plant of several species of Nymphalidae butterflies, known Volatile Collection Headspace collections were per- as passion vine butterflies (Brown and Mielke 1972; formed using glass chambers (11.5 9 35 cm), inside a Bianchi and Moreira 2005), which are common herbivores room with controlled temperature and humidity conditions of passion fruit plantations in Brazil (Rossetto et al. 1974). (24 ± 2 °C, 12 L: 12 D, 58 ± 2%). Samples were collected Heliconius erato phyllis (Fabricius 1775) (Lepidoptera: using a humidified and charcoal-filtered airflow at Nymphalidae) uses passion vines for oviposition and as a 0.5 L min−1 per chamber. VOCs were collected on glass larval food plant (Benson et al. 1976). Furthermore, studies columns containing 20 mg of the polymer HayeSep Q 80– suggested that Heliconiinae butterflies and its host plants 100 mesh (Althech, Lokeren, Belgium), and eluted with have coevolved. Evidence suggests that development of 240 μL of double-distilled HPLC-grade hexane (Zarbin plant defenses have affected diversification of both passion et al. 1999). An internal standard (IS) of heptadecane (C17: fruits and Heliconiinae butterflies (Smiley 1985a; Ehrlich 150 or 750 ng) was added to the final extract. Then, the and Raven 1964). Strategies used by passion vine plants final IS concentration was calculated and the extract was include structural modifications, which can discourage or quantified based on the IS peak area. For the purpose of prevent oviposition (Willlians and Gilbert 1981; Gilbert quantification accuracy, only peaks greater than 30 ng were 1982, 1991), perforate larvae (Gilbert 1971) and attract considered for the analyses. ants as predators (e.g., extrafloral nectaries) (Gilbert 1975; Passiflora edulis seedlings (five months of age and 10 Smiley 1985b). Also, induced responses to herbivory by leaves) were subjected to mechanical damage with a cir- Heliconiinae include the production of toxic and repellent cular pasta cutter (3 marked leaves), or to herbivory (10 compounds (Spencer 1988; Benson et al. 1976). Some of larvae), or to oviposition (five mated females that laid a the Heliconiinae responses to overcome plant defenses are mean of 20 eggs) by H. erato phyllis for 24 h or were used the preference to oviposit in the apex of tendrils avoiding as control plants. For the experiments, twelve plants were ant predation, the ability of larvae to coat trichomes with used once for each treatment. Treated plants were always silk to avoid cuticle perforation, and vision and memory compared to control plants. Mated females were identified abilities, which can help butterflies find and select host by the characteristic odor of the abdominal scent gland. plants (Benson et al. 1976). Headspace collections were performed at 24, 48, and 72 h Until now, induced defenses of Passiflora spp. have not after treatments for 24 h. been investigated concerning volatile emission or the Seedlings of each treatment were maintained in separate discrimination of different types of herbivore damage. For rooms to avoid plant-plant communication. Twelve repli- this reason, we aimed to compare the responses of P. cates were conducted of each experimental set. Each edulis to different injuries by identifying and quantifying experimental set contained four plants, three of them the VOCs emitted by treated plants (mechanical damage, treated (mechanical damage, herbivory, and oviposition) herbivory, and oviposition by H. erato phyllis) and control and one control, totaling 144 samples [12 replicates 9 4 plants. treatments = 48; 48 9 3 (24, 48, and 72 h) = 144]. Col- lections began at the same time each day until the end of the experiment. Extracts were stored at −20 °C until further Methodology analysis. VOCs were compared between treatments using gas chromatography (GC) and using combined gas chro- Rearing of Heliconius erato phyllis and cultivation of matography and mass spectrometry (GC–MS). Passiflora edulis seedlings Adults butterflies were collected Analytical Procedures Extracts were analyzed by com- in a native forest inside the Centro Polite´cnico, Universi- bined gas chromatography and mass spectrometry (GC– dade Federal do Parana´, in Curitiba, Parana´, Brazil. For the MS) on a Shimadzu QP 2010 Plus. The GC and GC/MS rearing and the manipulation of adults an insectary coated were equipped with a RTX-5 column (30 m 9 0.25 mm i. with Sombrite ®, which allows passage of 30% of direct d., 0.25 mm film thickness; Restek, Bellefonte, PA, USA). sunlight, was used as a semi-natural environment. Inside Injections of 1 μL were performed in the splitless mode, the insectary, pots with P. edulis; P. actinia Hooker, 1943; with an injector temperature of 250 °C. The column oven Impatiens walleriana Hook; Lantana camara Linnaeus, temperature was held at 50 °C for 1 min, increased to 250 ° 1753; and containers with artificial diet (Borges et al. 2010) Cat7°C min−1, and held for 10 min. Helium was the were used as oviposition and feeding substrate, respec- carrier gas at a linear velocity of 36.3 cm s−1. The same tively. Passiflora edulis individual seeds (Isla Pak®) were parameters were used for all analyses. cultivated inside plastic bags (15 cm 9 25 cm) containing Identification of Compounds VOCs were identified by untreated soil in a greenhouse. As seeds were purchased, Kovats indices (KI) and mass spectra comparisons with the 123 Terpenoids dominate the bouquet of volatile organic compounds produced by Passiflora edulis… 125 literature (Adams 2007; El-Sayed 2016), and by co-injec- variation (Fig. 3a). ANOVA showed that volatiles inter- tions with commercially available standards. 5-hydroxi-4- acted with treatment and time (Pillai trace = 0.041, octanone, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), far- F 6 = 2.261, P ≤ 0.5*), with treatment alone (Pillai nesene, and (3E,7E)-4,8,12-trimethyl-1,3,7,11- trace = 0.019, F 3 = 3.407, P ≤ 0.05*), and with time tridecatetraene (TMTT) were tentatively identified. Non- (Pillai trace = 4.73e-06, F 2 = 13.557, P ≤ 0.001*). In the anal, decanal, (E)-β-caryophyllene, (E)-β-ocimene, and plot of PCs (Fig. 3b), it is possible to see that volatiles (Z)-β-farnesene were identified by co-injections with indicated by X2, X4, X7, and X9 (EbOci, DMNT, EbCar, authentic samples (Sigma-Aldrich Chemical Company— TMTT) are separated from the other volatiles. They rep- Milwaukee, WI, USA). resent the increased emission of volatiles after 24 h of Statistical Analyses The bouquet of VOCs was analyzed herbivory (see Figs. 1, 4). Tukey tests showed the dis- by a Principal Components Analysis (PCA) followed by an tinction between HB plants and control plants (Fig. 4a) and Analysis of Variance (ANOVA). The ANOVA model between samples taken at 24 h from other experimental tested all possible interactions (volatiles, treatment, and times (Fig. 4b). time). Then, all nonsignificant effects were discarded and the best subset model was retained. Finally, a Tukey test was performed to test for differences between pairs of Discussion means. All tests were performed using R version 3.3.1 and R Studio version 0.99.903 (R Development Core Team VOCs emitted by healthy plants are usually modified fol- 2016). Discrepant samples of control plants were removed lowing mechanical damage and herbivore feeding. from the analyses. Specifically, terpenoids [e.g., (E)-β-ocimene, DMNT, far- nesane, (E)-β-caryophyllene, (Z)-β-farnesene, TMTT] are often emitted by plants in response to both wounding and Results herbivore attack (Walling 2000). In spite of that, our study showed no qualitative differences among treatments. VOCs of Passiflora edulis: identification, analysis, and However, ANOVA results indicated quantitative signifi- comparisons of treatments Nine compounds were quanti- cant differences between treatments, showing that samples fied and identified in the extracts of undamaged and collected at 24 h after herbivory were significantly different damaged P. edulis (Table 1). No qualitative differences from other samples. Treated plants clearly emitted greater among treatments were detected. amounts of VOCs after 24 h of damage, especially of ter- The terpenoid TMTT1 (9) showed the greatest increase penoids in MD and HB plants. MD-treated plants showed a of emission after 24 h of treatments when compared with general increase of VOCs after 24 h (EbOci, 10Al, and control: 6381 (HB), 397 (MD), and 224 (OV) times more. TMTT) and 48 h (Far and EbOci). HB plants showed a higher increase of terpenoids emission The quantitative variability in volatile emissions by (compounds 2, 4, 6–9) after 24 h of treatment when com- plants treated equally may be a result of plant genetic pared with OV, MD, and untreated plants (Table 1; Figs. 1, variation (Delphia et al. 2009; Dicke and Baldwin 2010), of 2). For example, P. edulis emitted 50, 46, 11, 6, and 3.6 physiological variation caused by biotic and abiotic stress, times more DMNT2 (4), (E)-β-ocimene, (Z)-β-farnesene, despite our efforts to maintain environmental and experi- (E)-β-caryophyllene, and farnesene, respectively, after 24 h mental stability (Oluwafemi et al. 2012; Kask et al. 2013; of herbivory, when compared with control plants. After Leppik et al. 2014; Niederbacher et al. 2015; Kariyat et al. herbivory, compounds 5HiOcta, EbOci, TMTT reached a 2017), and could also be affected by differences in her- peak of emission at 24 h (Fig. 2). Due to the high variation bivory damage (McCormick et al. 2014). In nature, the within treatment groups, chromatograms in Fig. 1 are variation in the emission of compounds by plants may representative. increase the number and the variability of predators MD plants showed a general increase of VOCs com- attracted to harmed or unharmed plants (Price et al. 1980). pared to untreated plants, usually reaching a peak of In the case of Passifloraceae plants, it remains unknown emission after 48 h (Far and EbOci), with the exception of about the predators and parasitoids attracted by volatiles EbOci, 9Al, DMNT, 10Al, and TMTT, which displayed a emitted after damage caused by H. erato phyllis. peak of emission at 24 h (Fig. 2). The increase in terpenoids emission (EbOci, DMNT, OV plants emitted (E)-β-ocimene in high amounts only Far, EbCar, EbFar, and TMTT) at 24 h after herbivory is after 72 h. Moreover, nonanal and (E)-β-caryophyllene evident (see Fig. 2 HB), which is represented in Fig. 4. reached a peak of emission at 24 h (Fig. 2). Terpenoids (e.g., α-pinene, limonene, linalool, ocimene, Statistical analyses indicated that the first principal caryophyllene, and farnesene) are usually released after component (PC1) of PCA explained 52.44% of the insect attack, especially 24 h after damage (Arimura et al. 123 126 E. de O. Borges et al.

2008; Heil 2008; War et al. 2011; Martins and Zarbin SE 1.76 1.58 1.02 0.55 2.10 3.13 27.14 3.62 0.56 )-4,8- ± 2013). For example, Eucalyptus benthamii Maiden & ± ± ± ± ± ± ± ± ± E ( Cambage (Myrtales: Myrtaceae) plants released greatest amounts of monoterpenes and sesquiterpenes after the DMNT attack by Thaumastocoris peregrinus Carpintero & Del- SE Mean 65.645.20 3.88 12.31 7.18 2.62 2.39 6.37 2.05 10.24 11.04 10.77 6.13 32.71 19.66 9.80 2.31 1.41

± lape´, 2006 (Heteroptera: Thaumastocoridae) (Martins and ± ± ± ± ± ± ± ± ± Zarbin 2013) and soybean plants (Glycine max (Linnaeus) Merrill (Fabales: Fabaceae)) emitted volatiles including camphene, myrcene, (E)-β-ocimene, MeSA, and TMTT SE Mean 82.6414.94 78.43 51.25 17.01 12.39 18.54 25.84 5.86 36.84 18.94 48.32 21.97 180.91 19.06 47.54 18.68 5.62 after the attack of bugs, which attracted the egg parasitoid, ± ± ± ± ± ± ± ± ± ± Telenomus podisi (Ashmead, 1893) (Hymenoptera: Sce- lionidae) (Moraes et al. 2009). (E)-β-ocimene is a monoterpene commonly found in the volatile blend emitted by leaves in response to herbivory SE Mean 162.05 231.28 0.761.15 43.52 0.30 165.71 0.90 29.14 18.57 37.31 8.93 74.51 9.85 79.76 510.54 0.98 29.45 and mechanical wounding (Pare´ and Tumlinson 1999; ± ± ± ± ± ± ± ± ± ± Martins et al. 2016). Moreover, Fatouros et al. (2012)

)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, β E reported the enhanced emission of (E)- -ocimene by ,7

E Brassica nigra Linnaeus (Brassicales: Brassicaceae) after SE Mean (3 17.74 265.89 2.420.19 5.58 0.87 1.15 5.34 0.88 15.76 7.99 8.88 25.16 9.36 15.48 18.82 1.85 1.99 24 h of oviposition by Pieris brassicae (Linnaeus, 1758) ± ± ± ± ± ± ± ± ± ± ) (Lepidoptera: Pieridae). Several studies indicate that air- TMTT

2015 borne (E)-β-ocimene emitted from plants can serve as a chemical cue, attracting parasitoids, predators, and polli- SE Mean 23.64 40.51 5.551.89 11.61 2.78 0.19 4.33 2.20 31.63 17.36 30.20 17.28 15.19 8.69 18.68 10.36 4.36 nator insects (Dudareva and Pichersky 2000; Pichersky and ± ± ± ± ± ± ± ± ± ± Gershenzon 2002). In our study, (E)-β-ocimene was emit- ; Braga et al.

plants from 24 to 72 h of the following treatments: mechanical damage, herbivory, and ted after oviposition and after herbivore and mechanical damage, with peaks of emission at 72 and at 24 h, 2011 SE Mean 12.32 40.70 0.972.71 24.29 0.45 1.89 0.66 9.23 13.00 15.38 65.64 7.252.15 25.87 19.72 0.65 15.36 respectively. This result indicates that (E)-β-ocimene was ± ± ± ± ± ± ± ± ± ±

ora edulis released after a short period (24 h) in response to damage, fl and after a longer period (72 h) after oviposition, sug-

Passi gesting different patterns of induction and release by the ; Macoris et al. SE Mean 4.625.14 19.11 5.06 4.78 3.60 8.39 8.81 1.60 83.72 5.91 23.96 8.965.38 20.31 9.47 0.64 0.88 damaged tissues. Furthermore, the exposure of plants to ± ± ± ± ± ± ± ± ± ± β 2009 (E)- -ocimene can activate the expression of defense

tentative identification based on mass spectra, genes, as in Arabidopsis thaliana (Linnaeus) Heynh t (Brassicales: Brassicaceae) (Fa¨ldt et al. 2003), through the

SE Mean release of genetically targeted signals by the emitter plant ) 33.497.42 14.36 5.99 18.90 5.76 14.24 2.99 9.25 13.89 19.09 101.95 8.6211.03 23.43 19.67 0.91 1.06 ± (Pontes et al. ± ± ± ± ± ± ± ± ± and its transportation, absorption, and perception by the 2016 receiver plant by means of VOCs receptors (Baldwin et al. 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h standard error; 2006). The increase of (E)-β-ocimene indicates that P. SE P. edulis SE) (ng) of VOCs emitted by SE Mean edulis plants might use this volatile as a cue to predators 3.070.79 62.89 1.26 30.97 0.26 16.02 1.41 20.11 8.50 12.75 4.53 27.92 0.05 22.42 31.78 0.79 1.19 ± ± ; El-Sayed ± ± ± ± ± ± ± ± ± and other plants to indicate the presence of herbivores.

2007 Butterflies often use chemical compounds obtained from Mean

(Lepidoptera: Nymphalidae) plants to produce their volatile signals (Boppre´ 1984;

891 2.21 Schulz et al. 2004). (E)-β-ocimene is a described anti- 10501106 4.92 1119 4.85 1206 3.24 1329 1.07 10.36 1435 13.00 14621583 6.79 0.08

t aphrodisiac compound of Heliconius melpomene (Lin- a naeus, 1758) (Lepidoptera: Nymphalidae), present in lower a a,b amounts in the abdominal scent glands of male valves, and t a a

t in Heliconius spp. Kluk, 1780 (Schulz et al. 2008; Estrada Kovats Indices (DB-5 column), t -caryophyllene -ocimene -farnesene β β β β

KI et al. 2011). For this reason, it is suggested that (E)- - )- )- )- E Z E nonanal DMNT decanal farnesane ( ( TMTT 5-hidroxi-4-octanone ( Heliconius erato phyllis ocimene (5) may be sequestered from P. edulis by Heli- conius larvae, including H. erato phyllis larvae, to compose Identification and quantification (mean the odoriferous bouquet of the abdominal scent glands 9Al DMNT 10Al Far EbCar EbFar TMTT 5HiOcta EbOci

peak number, located on the valves of adult males (Borges et al., Compounds present in the juice and fruit pulp of Identification based on co-injection with authentic samples 3 4 5 6 7 8 9 1 2 Table 1 No a b dimethyl-1,3,7-nonatriene and KI (Adams oviposition by No Compounds KI Control Mechanical damage (MD) Oviposition (OV)unpublished data). Oviposition (OV) 123 Terpenoids dominate the bouquet of volatile organic compounds produced by Passiflora edulis… 127

Fig. 1 Comparisons of the most representative chromatograms of Passiflora edulis plant extracts (24 h) treated with herbivory (HB) and oviposition (OV) by Heliconius erato phyllis, mechanical damage (MD), and control plants (C). HB chromatogram represents the VOCs (1–9) emitted by P. edulis plants. *contaminants and column bleed. VOCs: 1 (5- hidroxi-4-octanone), 3 (nonanal), 5 (decanal); DMNT: (E)-4,8-dimethyl-1,3,7- nonatriene; TMTT: (3E,7E)- 4,8,12-trimethyl-1,3,7,11- tridecatetraene

The homoterpenes DMNT and TMTT (respectively, their prey (Pare´ and Tumlinson 1999). Thus, the (E)-4,8-dimethyl-1,3,7-nonatriene and (3E,7E)-4,8,12-tri- sesquiterpenes (Z)-β-farnesene, (E)-β-farnesene, (Z,E)-α- methyl-1,3,7,11-tridecatetraene) are emitted by several farnesene, (E,E)-α-farnesene, and (E)-nerolidol were plants, including maize, lima bean, Arabidopsis, and emitted by in Pteris vittata Linnaeus (Polypodiales: Pteri- tomato (Hopke et al. 1994; Ament et al. 2004; Martins daceae) in response to herbivory of Spodoptera littoralis et al. 2016). TMTT has been reported as an induced (Boisduval, 1833) (Lepidoptera: Noctuidae) larvae (Im- volatile of cotton, Gossypium hirsutum (Hegde et al. 2011) biscuso et al. 2009). Genetically modified plants that (Malvales: Malvaceae), infested with Aphis gossypii Glo- constitutively express (E)-β-farnesene, which is the alarm ver, 1877 (Homoptera: Aphididae). Furthermore, plants of pheromone of many aphid species, not only repelled Myzus A. thaliana (Bruce et al. 2008) and G. max (Moraes et al. persicae (Sulzer, 1776) (Hemiptera: Aphididae) but also 2009) treated with cis-jasmone also displayed induced attracted parasitic wasps, such as Diaeretiella rapae emission of TMTT. In cotton fields, synthetic DMNT (M’Intosh, 1855) (Hymenoptera: Braconidae) (Beale et al. released by controlled dispersers attracted generalist 2006). predators belonging to different taxonomic orders of Following herbivory, farnesane was emitted by P. edulis insects (Yu et al. 2008). According to Chen et al. (2011), plants in small amounts as compared to other terpenoids. DMNT plays an important role in plant defense. Both However, significantly higher amounts of this compound tomato and Arabidopsis plants, for example, emit DMNT are present in headspace volatiles of bacterially infected that is subsequently used as a kairomone by predatory rice seedlings as compared to uninfected control plants mites (Kant et al. 2004; Kappers et al. 2005). (Sun et al. 2016). Farnesane has been also reported in (E)-β-caryophyllene has been reported to attract enemies ginseng seedlings grown in vitro and subsequently of both above- and below-ground herbivores in maize mechanically damaged or treated with methyl jasmonate (Kollner et al. 2008), and to be released by maize plants (MeJA) (Balusamy et al. 2015). attacked by larvae of Diabrotica virgifera virgifera This is the first study of volatiles emitted by a species in LeConte, 1868 (Coleoptera: Chrysomelidae) attracting the Passifloraceae. In spite of that, Jardim et al. (2010) entomopathogenic nematodes that infect and kill this root investigated the defense response of P. edulis against two pest (Degenhardt et al. 2009). Irmisch et al. (2014) lepidopteran species: a specialist [Agraulis vanillae vanil- described this compound as an herbivore-inducible volatile lae Boisduval & Le Conte, [1835] (Lepidoptera: in poplar. Nymphalidae)] and a generalist (Spodoptera frugiperda (J. (E)-β-farnesene has an important role in plant–insect E. Smith, 1797) (Lepidoptera: Noctuide)). In response to interactions (Crock et al. 1997). Insects release β-farnesene attack by both lepidopteran species, lipoxygenase activity as both pheromones and allomones, and many predatory was induced. That study focused on a key enzyme of the insects emit β-farnesene as a kairomone to locate and find jasmonic acid biosynthetic pathway (13-LOX), which is 123 128 E. de O. Borges et al.

Fig. 2 Comparisons of the VOCs emissions of Passiflora edulis plants (ng), after 24–72 h of treatments [herbivory (HB) and oviposition (OV) by Heliconius erato phyllis, mechanical damage (MD), and control plants]

Fig. 3 Results of the principal component analysis (PCA), which in the analysis; b Distribution of volatiles (X1–X9 in red) between tested the bouquet of VOCs emitted by control and treated (mechan- PC1 and PC2. Numbers in black represent all samples that were ical damage, herbivory and oviposition by Heliconius erato phyllis) analyzed. Axes represent PC values and the lower negative numbers Passiflora edulis plants. a Ordination of principal components. PCs indicate higher variance of X1–X9 above the red line (broken stick) are sufficient to explain the variance

123 Terpenoids dominate the bouquet of volatile organic compounds produced by Passiflora edulis… 129

Fig. 4 Results of ANOVA followed by a Tukey test, which tested the times (24, 48, and 72 h). Different letters indicate significant bouquet of VOCs emitted by Passiflora edulis plants. a comparisons differences. Y-axis correspond to PC1 values. A higher variance of treatments (control, mechanical damage (MD), herbivory (HB), creates lower negative numbers and oviposition (OV) by Heliconius erato phyllis); b comparisons of localized in plastids of passion fruit leaves, and is wound- plant volatile emission. Plant Physiol 146:965–973. doi:10.1104/ and MeJa-inducible (Rangel et al. 2002). Their results pp.107.111088 Arimura GI, Matsui K, Takabayashi J (2009) Chemical and molecular suggested that the response of P. edulis to herbivory could ecology of herbivore-induced plant volatiles: proximate factors be mediated by jasmonates. Furthermore, the effects of and their ultimate functions. Plant Cell Physiol 50:911–923. MeJa, mechanical wounding, and feeding by larvae of A. doi:10.1093/pcp/pcp030 vanillae vanillae upon trypsin inhibitory activity in passion Baldwin IT, Halitschke R, Paschold A, von Dahl CC, Preston CA (2006) Volatile signaling in plant-plant interactions: ‘‘Talking fruit leaves were also investigated (Botelho-Ju´nior et al. Trees’’ in the genomics era. Science 311:812 2008). All treatments caused an accumulation of trypsin Balusamy SRD, Rahimi S, Sukweenadhi J, Kim Y-J, Yang D-C inhibitors, which strongly inhibited trypsin-like digestive (2015) Exogenous methyl jasmonate prevents necrosis caused by enzymes from sugar cane stalk borer (Diatraea saccharalis mechanical wounding and increases terpenoid biosynthesis in Panax ginseng. Plant Cell Tiss Organ Cult 123:341–348. doi:10. (Fabricius, 1794) (Lepidoptera: Crambidae)) in vitro, sup- 1007/s11240-015-0838-8 porting a possible protective function of wound-inducible Beale MH, Birkett MA, Bruce TJA, Chamberlain K, Field LM, Huttly trypsin inhibitors against insect predation. AK, Martin JL, Parker R, Phillips AL, Pickett JA, Prosser IM, Although the potential of plant volatiles in the attraction Shewry PR, Smart LE, Wadhams LJ, Woodcock CM, Zhang Y (2006) Aphid alarm pheromone produced by transgenic plants of parasitoids in programs of pest control is known (Zarbin affects aphids and parasitoid behavior. Proc Natl Acad Sci USA et al. 2009) and that volatiles released after herbivory 27(103):10509–10513 selectively attract parasitoids (De Moraes et al. 1998), to Benson WW, Brown KSJ, Gilbert LE (1976) Coevolution of plants date, there are no predators described for H. erato phyllis. and herbivores: passion flower butterflies. 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