Phytochemistry 72 (2011) 601–609
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Phytochemistry
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Spatial and temporal patterns of floral scent emission in Dianthus inoxianus and electroantennographic responses of its hawkmoth pollinator ⇑ Francisco Balao a, , Javier Herrera a, Salvador Talavera a, Stefan Dötterl b a Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Apdo. 1095, E-41080 Sevilla, Spain b University of Bayreuth, Dept. Plant Systematics, D-95440 Bayreuth, Germany article info abstract
Article history: Scent emission is important in nocturnal pollination systems, and plant species pollinated by nocturnal Received 6 October 2010 insects often present characteristic odor compositions and temporal patterns of emission. We investi- Received in revised form 2 February 2011 gated the temporal (day/night; flower lifetime) and spatial (different flower parts, nectar) pattern of Available online 2 March 2011 flower scent emission in nocturnally pollinated Dianthus inoxianus, and determined which compounds elicit physiological responses on the antennae of the sphingid pollinator Hyles livornica. Keywords: The scent of D. inoxianus comprises 68 volatile compounds, but is dominated by aliphatic 2-ketones and Dianthus inoxianus sesquiterpenoids, which altogether make up 82% of collected volatiles. Several major and minor com- Caryophyllaceae pounds elicit electrophysiological responses in the antennae of H. livornica. Total odor emission does Flower scent Electroantennographic detection not vary along day and night hours, and neither does along the life of the flower. However, the proportion GC/MS of compounds eliciting physiological responses varies between day and night. All flower parts as well as GC/EAD nectar release volatiles. The scent of isolated flower parts is dominated by fatty acid derivatives, whereas Aliphatic 2-ketones nectar is dominated by benzenoids. Dissection (= damage) of flowers induced a ca. 20-fold increase in the Hyles livornica rate of emission of EAD-active volatiles, especially aliphatic 2-ketones. Nocturnal pollination We suggest that aliphatic 2-ketones might contribute to pollinator attraction in D. inoxianus, even Repellent though they have been attributed an insect repellent function in other plant species. We also hypothesize that the benzenoids in nectar may act as an honest signal (‘nectar guide’) for pollinators. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction On occasions, scent composition can vary among different flower parts, which can contribute to facilitate pollinator orienta- Flower scent is an ancient mode of communication between tion (Bergström et al., 1995; Dobson et al., 1996; Dötterl and flowering plants and their animal pollinators (Schiestl, 2010; Jürgens, 2005; Flamini et al., 2002; Knudsen and Tollsten, 1991; Raguso, 2008). Individual flowers can produce a few to more than Pichersky et al., 1994; Raguso and Pichersky, 1999). Also, temporal one hundred volatile substances in varying proportions (Knudsen patterns of scent release can be species-specific and often match and Gershenzon, 2006), and the overall number of compounds de- the pattern of activity of nocturnal visitors (Altenburger and Matile, tected in floral scent is huge (more than 1700 in 990 plant taxa; 1988; Effmert et al., 2005; Dötterl et al., 2005b; Hoballah et al., Knudsen et al., 2006). Combined with visual cues, floral scent en- 2005; Loughrin et al., 1991; Raguso et al., 2003). In addition to hances visitor attraction (Raguso, 2001), but constitutive or induced scent, flower pollination by moths and sphingids is associated with flower volatiles may also have a defensive function and act as repel- relatively high nectar volumes that are secreted at night (Baker, lents (De Moraes et al., 2001; Euler and Baldwin, 1996; Gatehouse, 1961). However, nectars rarely are simple sugary solutions and, in- 2002; Kessler and Baldwin, 2007; Pare and Tumlinson, 1999). Olfac- stead, frequently contain a broad spectrum of metabolites, includ- tory cues are particularly important for nocturnal visitors, to the ing volatiles, which may alter the probability of a flower being point that the flowers pollinated by moths often present a charac- visited by pollinators and non-pollinators (Adler, 2000; Baker and teristic scent composition, described as ‘white olfactory image’ Baker, 1975, 1983; Kessler and Baldwin, 2007; Raguso, 2004). (Dobson, 2006). This is dominated by acyclic terpene alcohols Many species of the diverse genus Dianthus (ca. 300 spp.) are (e.g., linalool, nerolidol, farnesol), along with aromatic (e.g., methyl pollinated by settling moths (mainly Geometridae, Noctuidae) benzoate, benzyl acetate) and nitrogen-bearing compounds. and sphingids (Jürgens, 2004; Jürgens et al., 2003). The flowers of Dianthus produce a wide array of volatile compounds, from benze- noids/phenylpropanoids (e.g., phenylacetaldehyde, benzyl acetate,
⇑ Corresponding author. Tel.: +34 954559887. methyl benzoate, eugenol) to terpenoids (e.g., (E)-b-ocimene, E-mail address: [email protected] (F. Balao). b-caryophyllene, linalool) and nitrogen-bearing compounds
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.02.001 602 F. Balao et al. / Phytochemistry 72 (2011) 601–609
(Dobson, 2006; Jürgens et al., 2003). A typical example of a noctur- variation were 2-tridecanone, 2-undecanone, b-caryophyllene, nally pollinated species of this genus is Dianthus inoxianus Gallego, and (E)-farnesol, which together explained, according to a SIMPER a perennial herb that occurs in the xerophytic scrub of fixed sand analysis, 67% of total variation observed between day and night dunes in the SW Iberian Peninsula. D. inoxianus is the dodecaploid samples. Three of these compounds were emitted in significantly cytotype of the D. broteri complex, a recently-radiated polyploid higher amounts at night (Wilcoxon signed rank test, n = 20; 2-tri- group of Iberian carnations (Balao et al., 2009, 2010). The flowers decanone: V = 46, p = 0.05; b-caryophyllene, V = 37, p = 0.02; of D. inoxianus are strongly protandrous: they release pollen when (E)-farnesol: V = 36, p = 0.05). young (usually during the first five days of anthesis), and become receptive when they are old (the last three days). Flowers are 2.3. Scents from isolated floral organs and nectar sweet-scented, open at night, secrete abundant nectar at the bot- tom of a very elongated (ca. 30 mm) calyx. Nectaries are situated 2.3.1. Quantity in the base of the stamens, at the point where the filaments join Some organs, like the petals or the calyx, emitted almost eight- the anthopore (i.e., the elongated stalk between the sepals and fold relative to the whole (undamaged) flower headspace. Summed the petals). up, the collected emission from dissected flower parts amounted to D. inoxianus flowers are pollinated almost solely (75 out of 88 2073 (1289–3361) ng/4 min of scent. From the whole flower, we col- visits in 5 years of field observations, i.e., 85%) by one nocturnal lected 114 (36–158) ng/4 min (median and range), although simply nectar-feeding hawkmoth species (Hyles livornica Esper; Lepidop- damaging the calyx (by slitting it with a pair of forceps) stimulated tera: Sphingidae; Balao, 2010). On the other hand, H. livornica is scent production dramatically (4440 (2574–4635) ng/4 min). The a rather generalist flower visitor that can be found foraging on same was observed when flower parts were dissected sequentially the flowers of many plant species (Johnson and Liltved, 1997; John- (Fig. 2). Nectar samples were also odorous, 3.3 (2.5–9.4) ng/4 min. son et al., 2002). The narrow pollinator spectrum of D. inoxianus prompted us to investigate whether a specific flower volatile com- 2.3.2. Composition position shapes this plant-pollinator relationship. The quantitative composition of scent varied significantly The goals of this study were (1) to investigated the spectrum of among different floral parts (ANOSIM, R = 0.70, p < 0.01), and volatile compounds in D. inoxianus flowers; (2) to explore the tem- non-metric multidimensional scaling showed four main scent poral pattern (day/night and through a flower’s lifespan) of scent groups on the basis of relative composition (Fig. 3). One group in- emission in order to verify whether this pattern (day/night) cluded scent from whole flowers, which had a profile characterized matches the period of maximum pollinator activity; (3) determine by aliphatic 2-ketones (e.g., 2-nonanone) and relatively high whether there are any among-organ (within-flower) differences in amounts of sesquiterpenoids (e.g., (E,E)-farnesol (E,E)-farnesyl ace- volatile release that might influence pollinator behavior; and (4) to tate, (E)-b-bergamotene). The group of calyx samples was rela- identify the volatiles in the floral scent of D. inoxianus that H. livor- tively rich in lipoxygenase products or ‘green leaf volatiles’ (e.g., nica can perceive by olfaction. (Z)-3-hexenal, (Z)-3-hexen-1-ol). The group of samples from petals, stamens, gynoecia, and anthophores was characterized by high lev- 2. Results els of aliphatic 2-ketones (2-tridecanone, 2-undecanone and 2- pentadecanone). Lastly, nectar scent was relatively rich (56.8% of 2.1. Composition and quantity of floral scent total emission) in benzenoid compounds (benzaldehyde, benzyl alcohol, phenylacetaldehyde, 2-phenylethanol) and aliphatic 2-ke- The scent of D. inoxianus flowers included a total of 68 volatiles, tones (36.6%). Benzenoids were largely absent from whole-flower even though only 13 compounds attained percentages >1% of the samples (Table 1, Fig. 3), but were present in small amounts (often volatiles collected (Table 1). It was strongly dominated by a few less than 0.6%) in scent collected from individual floral parts. aliphatic ketones and sesquiterpenoids that together represented 82% of the total scent. The most abundant compounds (>7–10% of total emission during the day or night) were the aliphatic 2-ke- 2.4. Electrophysiologically active compounds tones 2-nonanone, 2-undecanone, and 2-tridecanone, and the ses- quiterpenoids (E)-b-bergamotene and (E,E)-farnesol. Minor scent The antennae of male and female H. livornica responded to all components included the fatty acid derivatives (Z)-3-hexenyl ace- (quantitatively) dominant scent compounds in the GC–EAD exper- tate and 2-pentadecanone, and the sesquiterpenes b-caryophyl- iments. Responses were elicited in both sexes (at least in three lene, a-caryophyllene, (E,E)-a-farnesene, and (E)-nerolidol. male and three female antennae) by 2-nonanone, 2-undecanone, 2-tridecanone, 2-pentadecanone, b-caryophyllene, (E)-b-bergamo- 2.2. Temporal patterns in scent emission tene, (E,E)-a-farnesene, (E)-nerolidol, (E,E)-farnesol, and an un- known oxygenated farnesene derivative (Fig. 4, Table 1). Scent emission began as soon as the flowers opened but total Although average diurnal and nocturnal total amount emissions daily scent emission was neither affected by flower aging (Fig. 1; were statistically undistinguishable (see Section 2.2.), in the statis- tical analysis the antennal activity showed a strong interaction be- non-parametric Kruskal–Wallis test: H4,20 = 5.18, p = 0.27) nor sex- tween time of emission (day vs. night) and scent composition ual stage (H9,20 = 15.9, p = 0.07). With regard to the absolute amount of each compound, significant differences were detected (paired ANOVA, F1,36 = 31.63, p < 0.001). At night, the scent was among plants (ANOSIM, R = 0.52, p < 0.001) and moderate, but still rich in active compounds such as 2-tridecanone and (E,E)-farnesol, significant, differences among sexual stages (ANOSIM, R = 0.18, but relatively poor in non-active volatiles (Fig. 5). The opposite pat- p = 0.025). In contrast, the relative composition of scent did not tern was observed during daytime. vary among plants (ANOSIM, R = 0.02, p = 0.35) or sexual stages (ANOSIM, R = 0.06, p = 0.19). 3. Discussion Average diurnal and nocturnal total emissions were statistically undistinguishable (Wilcoxon signed rank test, n = 20, V = 76, 3.1. Floral scent: composition and timing p = 0.46), and so were the global proportions of compounds (ANO- SIM, R = 0.11, p = 0.26). However, there was some variation be- In contrast with most plant species pollinated at night, which tween day and night samples, and most responsible for this are odorous basically at night (Altenburger and Matile, 1988; Effm- Table 1 Relative amounts of volatile compounds detected in Dianthus inoxianus floral scent. KI is the Kovats retention index. Compounds eliciting electrophysiological antennal responses (EAD-active) in Hyles livornica are marked with an ⁄.
Compound KI EAD-active Daily emission Floral parts emission Day N = 20 Night N = 20 Flower N = 3 Calyx N = 3 Petals N = 3 Anthers N = 3 Ovary N = 3 Anthophore N = 3 Nectar N =3 Benzenoids Benzaldehyde a 980 – – – – – – – – 24.84 Benzyl alcohola 1048 – – – – 0.05 0.23 – – 1.54 Phenylacetaldehydea 1060 – – 0.01 1.58 0.01 – – 0.04 29.60 2-Phenylethanola 1129 – – – 0.06 0.08 0.43 – 0.16 0.77 (Z)-3-Hexenyl benzoate 1580 – – – – 0.01 0.01 – – – Benzyl benzoatea 1787 – – – – – – 0.11 – – Fatty acid derivatives (Z)-3-Hexenal 811 1.42 – 1.65 11.04 – – – – – (Z)-3-Hexen-1-ola 876 7.63 1.21 4.17 34.13 0.73 1.58 0.12 2.87 – 2-Heptanone 895 0.49 0.55 0.76 – – – – – – (E,E)-2,4-Hexadienal 934 3.48 – 0.29 4.01 – – – – – (Z)-3-Hexenyl acetatea 1016 7.19 0.53 0.65 1.40 – – 1.61 – – 2-Nonanonea 1097 ⁄ 8.71 9.61 13.54 0.97 0.88 0.90 1.09 2.78 – (Z)-3-Hexenyl butyratea 1188 0.72 0.22 – 0.11 – – – – – a 1196 0.15 0.05 – – – – – – 0.11
2-Decanone 601–609 (2011) 72 Phytochemistry / al. et Balao F. Branched C11 2-ketone 1286 – – – – 0.05 – – – – 2-Undecanonea 1294 ⁄ 17.34 18.96 6.89 16.85 21.89 6.97 10.97 20.82 6.97 2-Undecanol 1301 – – – 0.36 0.86 0.56 1.09 – – Branched C12 2-ketone 1367 – – – 0.08 0.14 0.55 0.20 – – 2-Dodecanonea 1394 – – – 0.04 0.10 0.17 0.11 0.15 – m/z: 43,55,41,69,83,39 1428 – – – 0.03 0.08 0.09 0.10 – – Branched C13 2-ketone 1461 – – – 0.12 0.12 0.77 0.49 – – 2-Tridecanonea 1497 ⁄ 18.92 25.30 8.83 23.73 66.24 76.03 76.37 70.93 27.62 Tridecanol 1500 – – – 0.07 0.41 0.92 0.77 – – Branched C15 2-ketone 1660 – – – – 0.15 0.20 0.31 – – 2-Pentadecanonea 1697 3.38 4.28 2.25 0.61 3.50 6.44 5.38 1.30 1.89 2-Heptadecanone 1898 – 0.08 – – – 0.06 – – – Monoterpenoids (E)-b-Ocimenea 1056 – – – – – 0.04 – – – Linaloola 1105 0.35 0.13 – – – – – – – Unknownsb 0.052 0.062 –––––– – m/z: 43,67,79,81,39,107 1377 – – 0.62 0.11 0.06 0.50 0.15 – 0.59 Sesquiterpenoids b-Caryophyllenea 1450 ⁄ 1.57 5.40 0.26 3.75 0.79 0.36 0.26 0.94 1.41 (E)-b-Bergamotene 1461 ⁄ 8.54 9.24 5.53 0.03 0.89 – 0.33 – – a-Caryophyllene a 1480 0.34 0.26 – 0.06 0.07 0.09 0.03 – 0.37 (E,E)-a-Farnesene a 1509 ⁄ 3.68 4.03 2.99 0.23 0.27 0.50 0.12 – – m/z: 119,91,105,93,77,39 1518 0.78 0.67 0.60 – 0.04 – – – – (E)-Nerolidola 1568 ⁄ 0.70 0.79 0.60 trace 0.28 0.39 0.40 – – (E,E)-Farnesola,c 1728 ⁄ 8.78 13.09 36.71 – 1.35 0.18 – – – Oxygenated farnese derivative 1750 ⁄ 2.17 2.59 4.05 – 0.56 0.11 – – 0.12 m/z: 55,93,91,77,39,67 1773 – – – 0.20 – 0.76 – – – (E,E)-Farnesyl acetate 1842 0.42 0.03 8.05 0.32 – 1.07 – – 4.15 Unknownsb 3.1522 2.9323 1.5210 0.103 0.4416 0.097 –– – Unknowns m/z: 40,159,97,67,105,131 1592 0.03 0.02 – – – – – – –
a The identity was confirmed by synthetic compounds. b Unknown with a percentage amount of less than 0.5 were pooled with the superscript digit giving the number of pooled compounds. c This peak also contained small amounts of (E,E)-farnesal. 603 604 F. Balao et al. / Phytochemistry 72 (2011) 601–609
its isolated floral parts (Fig. 2). Seemingly, dissection stimulated scent emission, which suggests that damage caused naturally to flowers (e.g., by florivores) could also result in increased total vola- tile release. As for the composition of scent, that of isolated flower parts was dominated by ketones in all cases except the calyx (Fig. 3), which was relatively rich in lipoxygenase products or ‘green leaf volatiles’ (e.g., (Z)-3-hexenal, (Z)-3-hexen-1-ol). The fact that calyces released ‘green leaf volatiles’ after dissection is not surpris- ing, as these compounds are commonly emitted by damaged leaves and calyces in other species (D’Auria et al., 2002; Dötterl and Jür- gens, 2005; Loughrin et al., 1994; Pare and Tumlinson, 1999; van Poecke et al., 2001). The rest of D. inoxianus floral organs were all very similarly scented, which contrasts with findings from many other species (Bergström et al., 1995; Dobson et al., 1996; Flamini et al., 2002; Knudsen and Tollsten, 1991; Pichersky et al., 1994; Ra- guso and Pichersky, 1999). It has been hypothesized that within- flower variations in scent can contribute to facilitate pollinator ori- entation (see references above), but in the case of D. inoxianus this Fig. 1. Total scent emission along the lifespan of five flowers from different D. inoxianus plants. Horizontal lines represent the median, and boxes and whiskers can only apply to the odorous nectar, which differed from the other the interquartile range and the non-outlier ranges, respectively. flower parts in its relative scent composition. Interestingly, in a spe- cies of the related genus Silene, it has been demonstrated that petals ert et al., 2005; Dötterl et al., 2005b; Hoballah et al., 2005; Loughrin and the anthophore (which contains the nectaries) emit different et al., 1991; Raguso et al., 2003), the flowers of D. inoxianus emitted volatiles (Dötterl and Jürgens, 2005). scent continuously during the night and the day, and throughout the period of anthesis. Nevertheless, nocturnal scent was enriched 3.1.2. The hidden, odorous nectar of D. inoxianus in specific compounds such as 2-tridecanone, b-caryophyllene, and The nectar of D. inoxianus had a distinctive scent dominated by (E)-farnesol. Shifts in scent composition between day and night aliphatic 2-ketones as well as by benzenoids (Table 1, Fig. 3). While have been reported in other species as well (Baldwin et al., 1997; some of these compounds (e.g., aliphatic 2-ketones) could accumu- Schiestl et al., 1997) and mechanistic hypotheses based on light late in nectar by passive diffusion from floral organs, where they regulation have been put forward to explain the changes. For were abundantly emitted, the presence of benzenoids cannot be example, the enzymatic pathways could be light-regulated, or explained on this basis, as they were largely absent from the rest compound synthesis might become partially deactivated during of the flower. Therefore, it is more likely that benzenoids were ac- daytime (Hendel-Rahmanim et al., 2007; Simkin et al., 2004; Ver- tively released in the nectaries. Previous studies have demon- donk et al., 2003). This has been demonstrated in Lycopersicon strated the effectiveness of benzenoids in attracting moths hirsutum f. glabratum, a species in which the concentration of 2-tri- (Dötterl et al., 2006; Haynes et al., 1991; Heath et al., 1992; Huber decanone (the same substance that dominates the scent of D. inox- et al., 2005). Because of the differential emission of benzenoids ianus, and that is enriched at night) follows both light intensity and from the nectar in D. inoxianus, these compounds could act simi- day length (Kennedy et al., 1981), suggesting that this might occur larly to a nectar guide (Dötterl and Jürgens, 2005; Howell and Ala- in D. inoxianus as well. rcón, 2007) and/or indicate rewarding flowers (‘honest signal’; Heinrich, 1979; Holder and Lewis, 2003; Marden, 1984; Raguso, 3.1.1. Intrafloral variations in scent 2004). In addition, the attested antifungal (Wang et al., 2005) The amount of volatiles released by a whole, intact D. inoxianus and antibacterial (Prabuseenivasan et al., 2006) properties of benz- flower was significantly lower than the sum of volatiles emitted by enoids could help to control microbial growth in nectar.
Fig. 2. Volatile emission from D. inoxianus flowers, their isolated floral parts, and nectar samples. Bars show the median and upper range, n = 3 in all cases. F. Balao et al. / Phytochemistry 72 (2011) 601–609 605
Fig. 3. Non-metric multidimensional scaling (NMDS) ordination (based on Bray–Curtis scent similarities) of three D. inoxianus flowers (each one from a different plant), their dissected floral parts, and nectar.
Fig. 4. GC–EAD recordings of H. livornica antenna exposed to the volatile compounds of D. inoxianus given as the average responses of the 12 separate runs performed. Numbers indicate EAD responses: 2-nonanone (1), 2-undecanone (2), b-caryophyllene and (E)-b-bergamotene (3), 2-tridecanone (4), (E,E)-a–farnesene (5), (E)-nerolidol (6), 2-pentadecanone (7), (E,E)-farnesol (+ small amounts of (E,E)-farnesal) (8) and oxygenated farnesene derivative (9). The latter compound (9) elicited an antennal response in 6 of the 12 runs, but an obvious response is not visible in the plotted averaged line. The response marked with ‘‘⁄’’ was to nonanal, which occurred also in the ambient control samples.
3.2. The physiological response of H. livornica to D. inoxianus scent been identified in other hawkmoth-pollinated Dianthus (e.g., Dian- thus arenarius, Dianthus superbus and Dianthus monpessulanus; Jür- Benzenoids were not tested for electrophysiological activity on gens et al., 2003), in nocturnally pollinated species of the related the antennae of the main pollinator since they were only present genus Silene (Jürgens et al., 2002), as well as in sphingophilous spe- in the nectar but not in the total flower headspaces samples. Our cies of Caprifoliaceae, Amaryllidaceae, Solanaceae and Nyctagina- GC–EAD experiments demonstrated that both the male and female ceae (Levin et al., 2001, 2003; Miyake et al., 1998). In addition, the individuals of H. livornica can sense several of the compounds emit- sesquiterpenes that elicited antennal responses in H. livornica do ted by D. inoxianus, i.e., aliphatic 2-ketones and sesquiterpenoids. so in other hawkmoth species (e.g., Manduca sexta, Fraser et al., Some of these EAD-active compounds, especially sesquiterpenoids 2003; Sphinx perelegans, Raguso and Light, 1998; and Hyles lineata, (e.g., a-and b-caryophyllene, (E)-nerolidol, (E,E)-farnesol) have also Raguso et al., 1996), which is in agreement with a role of these com- 606 F. Balao et al. / Phytochemistry 72 (2011) 601–609
tones is simply outbalanced by the presence of attractive compounds in the floral scent (e.g., EAD-active sesquiterpenoids like (E,E)-farnesol, (E)-b-bergamotene, (E)-nerolidol). Overall, the effect of aliphatic 2-ketones on the hawkmoth H. livornica (repel- lent vs. attractive cue), and their role in the pollination of D. inox- ianus (florivore defence vs. pollinator attraction) is a complex subject that deserves further study.
4. Concluding remarks
The daily pattern of scent emission in D. inoxianus is unusual in that overall amount of volatiles released is not correlated with the period of activity of the major hawkmoth pollinator, H. livornica. However, the proportion of EAD-active compounds increases at night, matching the activity of the pollinator. The scent of nectar is dominated by benzenoids, which could act as a nectar guide and/or indicate rewarding flowers (‘honest signal’). In a response typical of plant defensive compounds, aliphatic 2-ketones in the floral scent increase 20-fold following flower damage; while these compounds have a deterrent function in other plant species, they might act as a cue to pollinators in D. inoxianus. Fig. 5. Relative proportions of EAD-active vs. non-active compounds during day and night. Horizontal lines represent the median, and boxes and whiskers the interquartile range and the non-outlier ranges, respectively, n = 19 in all cases. 5. Experimental pounds in pollinator attraction. However, it must be emphasized 5.1. Plant and insect material that aliphatic 2-ketones (2-tridecanone, 2-undecanone and 2-nona- none), to which H. livornica antennae responded, had not been re- Plants of D. inoxianus were cultivated from seeds in a green- ported before to date to elicit hawkmoth antennal responses. house of the University of Bayreuth and placed in a bed outside when they were three months old. At the onset of flowering, plants 3.3. Floral scent: defence or pollinator attraction? were placed back inside the greenhouse to prevent insect visitation to the flowers. Preliminary bioassays conducted in the field to determine the Individuals of H. livornica were reared from pupae obtained behavioral attractiveness of D. inoxianus scent using synthetic from The Lepidoptera Breeders Association (LBA, UK). The condi- blends of aliphatic 2-ketones and farnesol were inconclusive, as tions of rearing were a photoperiod of 18 h light and 6 h dark, H. livornica hawkmoths did not approach an experimental setup and a temperature of 26 °C (in light) and 18 °C (in darkness). with artificial flowers (odorous vs. negative controls; F. Balao, unpublished data). More detailed behavioral experiments are nec- 5.2. Volatile collection essary to establish the function of the aliphatic 2-ketones and ses- quiterpenoids in D. inoxianus, but preliminary wind-tunnel Floral scent was collected for three separate purposes: (1) to experiments with H. livornica indicated that the insects are effec- determine the scent emitted at daytime and at night during the tively attracted to the scent of natural flowers (four out of six indi- 7(9) days of floral anthesis; (2) to determine the scent emitted viduals responded positively; S. Dötterl, unpublished data). This, by different flower parts and by nectar; and (3) to obtain scent together with the fact that EAD-active compounds significantly samples for electrophysiological measurements. prevailed at night (matching the period of activity of H. livornica) ChromatoProbe quartz micro-vials of Varian Inc. (length: 15 mm; rather than during the day, suggests that scent as a whole has a rel- inner diameter: 2 mm) were cut at the closed end, filled with a mix- evant role in pollinator attraction. ture (1:1) of 1.5 mg Tenax-TA (mesh 60–80; Supelco, Bellefonte, The aliphatic 2-ketones that dominated the scent of D. inoxianus Pennsylvania, USA) and 1.5 mg Carbotrap B (mesh 20–40, Supelco, are hitherto not widely recognized as pollinator attractants. In- Bellefonte, Pennsylvania, USA), and used as adsorbent tubes. The stead, they are known to act as repellents/insecticides in several adsorbents were fixed in the tubes using glass wool. Simultaneous plant species (Dobson et al., 1999; Juvik et al., 1982; Lin et al., collections of the surrounding air were performed to distinguish be- 1987; Williams et al., 1980). They also appear in defensive secre- tween floral compounds and ambient contaminants. tions of insect larvae (Attygalle et al., 1993; Eisner et al., 1972; D. inoxianus individuals were sampled in the laboratory under Weatherston et al., 1979). In D. inoxianus the emission of aliphatic an extractor hood using a dynamic headspace method described 2-ketones is triggered by experimental damage to the flowers before (Dötterl et al., 2005b). A single flower was enclosed in situ Ò (causing a 20-fold increase), in a response typical of plant defen- within a polyester oven bag (10 cm  7 cm, Toppits ) and the sive compounds (Dicke and Hilker, 2003; Gatehouse, 2002; Van emitted volatiles were trapped in an adsorbent tube through the Poecke et al., 2001) suggesting that aliphatic 2-ketones may have use of a membrane pump (G12 01/EB; ASF Thomas, Inc.). The flow (in general) a repellent action in D. inoxianus. However, our find- rate was adjusted to 200 ml/min using a power supply and a flow ings indicate that these compounds might be either neutral or flo- meter. Samples were collected for 4 min (2 min bagging + 2 min ral attractants to the major pollinator, H. livornica. If the latter is collecting), a period which, from previous tests, was judged ade- the case then aliphatic 2-ketones would be acting like a floral filter quate to gather enough amount of compounds for the analyses. (Johnson et al., 2006; Junker and Blüthgen, 2010; Raguso, 2008) contributing to narrowing down the spectrum of pollinators to H. 5.2.1. Temporal pattern livornica (by repelling co-occurring potential pollinators). Alterna- Diurnal and nocturnal emission of scent was established from tively, it is also possible that the repellent action of aliphatic 2-ke- samples collected from five flowers of different D. inoxianus indi- F. Balao et al. / Phytochemistry 72 (2011) 601–609 607 viduals. Flowers were sampled at two-days intervals during the used to determined the total amount of each compound available 7(9) days of anthesis, once at midday (12:00) and another just after in the samples (Dötterl et al., 2005b). dusk (21:30, the peak of pollinator visitation in the field; Balao, unpublished data). Each time a flower was sampled its sexual stage 5.4. Electrophysiological analysis was recorded, differentiating eight sexual stages on the basis of the number of exerted pairs of stamens (stages 1–5) or style elongation Based on the same setup and method described by Dötterl et al. (initial, medium, and maximum, which correspond respectively to (2005a), gas chromatography coupled to electroantennographic stages 6, 7, and 8). detection (GC–EAD) was applied to identify which of the com- pounds emitted from D. inoxianus were perceived by the antennae 5.2.2. Flower parts sampling of two gravid females and three males of H. livornica. Both anten- To determine the scent emitted from different flower parts, we nae of the same individual were used for the analyses of different collected scent for 4 min from three in situ flowers of different indi- samples. One GC–EAD run was performed per antenna, except for viduals in the male stage (with six to eight anthers exerted). Then, two antennae which allowed two runs to be performed. In total 12 single flower parts (calyx, petals, stamens, gynoecium, and antho- runs were conducted. phore) were sequentially removed and separately put into differ- The GC–EAD system consisted of a gas chromatograph (Vega ent bags for scent collection for another 4 min. 6000 Series 2, Carlo Erba, Rodano, Italy) equipped with a flame ion- ization detector (FID), and an EAD setup (heated transfer line, 2- channel USB acquisition controller) provided by Syntech (Hilver- 5.2.3. Nectar sampling sum, Netherlands). A volume of 1 ll of the acetone-scent sample In order to collect nectar scent, we extracted 2.5–6 ll of nectar was injected splitless at 60 °C, followed by opening the split vent with 5 ll ‘Pyrex’ capillaries (Corning, New Cork, USA) from three to after 1 min and heating the oven at a rate of 10 °C minÀ1 to five flowers on each of three different plants. Each nectar sample 200 °C. The final temperature was held for 5 min. For the analyses, was deposited on filter paper, enclosed for 30 min within oven the ZB-5 column (length 30 m, inner diameter 0.32 mm, film thick- bags (10  7 cm), and the emitted volatiles trapped for 2 min fol- ness 0.25 lm, Phenomenex) was split at the end by the four-arm lowing the same procedure described above. flow splitter GRAPHPACK 3D/2 (Gerstel, Mülheim, Germany) into two deactivated capillaries (length 50 cm, inner diameter 5.2.4. EAD volatile sampling 0.32 mm) leading to the FID and EAD setup. Makeup gas (He, To obtain a scent sample from a flower (in male stage) for EAD 16 ml minÀ1) was introduced through the fourth arm of the studies, a slightly different dynamic headspace method was used: splitter. the flower was enclosed in situ in the same type of oven bags For the EAD, both sides of an excised antenna were plugged into (10 cm  7 cm), and the emitted volatiles were trapped overnight glass micro-capillary electrodes filled with insect ringer solution in large adsorbent tubes filled with 15 mg of Tenax-TA (mesh À1 À1 À1 (8.0 g l NaCl, 0.4 g l KCl, 4 g l CaCl2) and connected to silver 60–80) and 15 mg of Carbotrap B (mesh 20–40). Volatiles were wires. To identify the compounds eliciting signals in the insect eluted with 70 ll of acetone (SupraSolv, Merck KgaA, Germany) antennae, 1 ll of the acetone floral scent samples was placed in a and stored in a freezer at À80 °C for later use. quartz vial in the injector port of the Varian 3800 GC by means of the ChromatoProbe, and then analyzed by the GC–MS as de- 5.3. Chemical analyses of floral scent scribed above. Peaks eliciting EAD responses were located in the GC–MS runs by comparing the overall patterns of the MS- and The composition of the floral scent was analyzed on a Varian FID-chromatogram and by using the Kovats retention index (series Saturn 3800 gas chromatograph (GC) fitted with a 1079 injector of n-alkanes were injected in both systems). and a ZB-5 column (5% phenyl polysiloxane, length 60 m, inner diameter 0.25 mm, film thickness 0.25 lm, Phenomenex; Torrance, 5.5. Statistical analyses CA, USA), and a Varian Saturn 2000 mass spectrometer (MS) (Var- ian Inc., Palo Alto, CA, USA). The adsorbent tubes were inserted via Differences in the total absolute amount of scent both among Varians Chromatoprobe into the GC injector (Amirav and Dagan, days and among floral stages were evaluated using nonparametric 1997). The injector split vent was opened and the injector was Kruskal–Wallis rank sum test (K–W). Differences among time of heated at 40 °C to flush any air from the system. After 2 min the sampling (i.e., day vs. night emission) were tested with a paired split vent was closed and the injector heated at 200 °C minÀ1, then Wilcoxon signed rank test. Both test were performed with R statis- held at 200 °C for 4.2 min, after which the split vent was opened tical software ver 2.10.1 (R Development Core Team, 2009). and the injector cooled down. Electronic flow control was used To study variations in floral scent composition (in terms of both to maintain a constant helium carrier gas flow rate (1.8 ml minÀ1). absolute and percent amounts of each compound), sample-based The GC oven temperature was held for 7 min at 40 °C, then in- Bray–Curtis similarity indexes were computed. Similarities were creased by 6 °C minÀ1 to 260 °C and held for 1 min at this temper- subject to multivariate non-parametric analysis (ANOSIM; Clarke, ature. The mass spectra were taken at 70 eV with a scanning speed 1993) with 10,000 permutations, and flower age/sexual stage and of 1 scan sÀ1 from m/z 30–350. time of day (day/night) as factors nested within plant identity. A Data analyses were performed using the Saturn Software pack- SIMPER analysis was used to determine the mean dissimilarity in age 5.2.1. To identify floral scent compounds based on the GC-MS, scent between day and night samples and also to determine the the data bases NIST 02 and MassFinder 3 were used. Identifications compounds most responsible for variation in scent between day were confirmed by comparison of retention times with published and night. When performing multiple comparisons, the signifi- data (Adams, 1995). When available, identifications were con- cances of tests were Bonferroni-corrected. Both multivariate anal- firmed by comparing the mass spectra and retention times with yses were performed in using PRIMER 6 software (Clarke and those of authentic standards. We estimated total scent emission Gorley, 2006). (absolute amount; not for the sample collected for electrophysio- To assess whether the overall quantity of the electroantenno- logical analyses) by injecting known amounts of monoterpenoids, graphically active and non-active compounds varied between day benzenoids, and fatty acid derivatives via Varians Chromatoprobe. and night, we performed a two-way repeated measures ANOVA The mean response of these compounds (mean peak area) was that had activity (in the EAD) and time of sampling (day/night) 608 F. Balao et al. / Phytochemistry 72 (2011) 601–609 as factors in a crossed design: for each pair of samples (day vs. Dötterl, S., Füssel, U., Jürgens, A., Aas, G., 2005a. 1,4-Dimethoxybenzene, a floral night), compounds were categorized either as active or non-active, scent compound in willows that attracts an oligolectic bee. J. Chem. Ecol. 31, 2993–2998. the fraction of each type computed, and standardized to 100 (with- Dötterl, S., Wolfe, L.M., Jürgens, A., 2005b. Qualitative and quantitative analyses of in sample pairs). Computations were performed with STATISTICA flower scent in Silene latifolia. Phytochemistry 66, 203–213. 5.0 (StatSoft Inc., 1995). Dötterl, S., Jürgens, A., Seifert, K., Laube, T., Weibbecker, B., Schütz, S., 2006. Nursery pollination by a moth in Silene latifolia: the role of odours in eliciting antennal To test for differences in scent composition among flower parts and behavioural responses. New Phytol. 169, 707–718. (including nectar), and visualize these differences we built a Bray– Effmert, U., Grosse, J.R., Ursula, S.R., Ehrig, F., Kagi, R., Piechulla, B., 2005. Volatile Curtis distance matrix, based on the percent amount of com- composition, emission pattern, and localization of floral scent emission in Mirabilis jalapa (Nyctaginaceae). Am. J. Bot. 92, 2–12. pounds, which was then subject to ANOSIM analysis and non-met- Eisner, T., Kluge, A.F., Carrel, J.C., Meinwald, J., 1972. 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