Volatile organic compounds and host-plant specialization in European corn borer E and Z pheromone races

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VOCs collection

Maize field VOCs were collected by placing SPME fibers in open-air condition in the middle of the maize field. The fiber was attached at the top part of a maize stem. The forest VOCs were collected by placing SPME fibers in open-air conditions on the lower branches of trees at 1.50 m above the ground. The sampling was conducted at least 5 meters inside of the forest.

SPME fiber

SPME fibers DVB/CARBOXEN/PDMS 50/30 $m (Supelco) were conditioned by heating in the gas chromatograph injector at 250 °C for 5·min with helium as carrier flow. Cleaned fibers were then wrapped in aluminum foil and stored in individual screw-capped Pyrex glass tubes until use.

Chemical analysis

After volatile collection, SPME fibers were desorbed in a Varian 3400 GC injector held at 250°C. The GC was coupled to a MS detector Varian QIMS. Compound separation was carried out using a Rxi-5ms column (Restek, France) 30 m % 0.32 mm i.d., film thickness 1.0 $m). The column was programmed from 50 °C for after 3 minutes at 8 °C/min to 300 °C. Helium was used as carrier gas. Mass spectra were obtained in electron impact mode (70 eV) with the ion source at 230 °C. Compounds eluted from SPME collections were identified according to their mass spectra and retention index (RI). RIs were computed using C10 to C24 n-alkanes, eluted under the same conditions as the samples. Every compound spectrum and RI was compared with the RI and spectrum of standard and NIST 1998 library as reference using deconvolution software AMDIS32. The calibration curves of green leaf volatiles (GLV), homoterpenes (HT), monoterpenes (MT) and sesquiterpenes (SQT) were obtained by injection of standards: !-linalool, !-pinene, !-humulene, methyl salicylate (MeSA), cis-3-hexenol (Sigma-Aldrich), « -farnesene (Chemtech), ocimene (Fluka), cis-3-hexenyl acetate (Lancaster) and (E)-4,8-dimethyl-1,3,7- nonatriene (DMNT) (gift from Jarmo K.

Data analysis

All the raw GC-MS data of VOCs peak areas were transformed into nanograms (ng) using the calibration curves. Relative amounts of each compound was calculated by dividing the compound amounts (ng) by the sum of detected compounds amount of the same analyze and expressed as a percentage. Since there were seven replicates of VOCs profiles taken from the maize field and four from the forest landscape; percentage composition of each product is the mean ± standard error.

Maize headspace VOC

From maize headspace VOC collections, 21 components were detected and identified based on comparing the RI and mass spectra to authentic samples or appropriate data bases (Table 1a). Nineteen components out of 21 had previously been identified. Two VOCs were newly identified as maize VOCs: p-cymene, and a compound tentatively identified as selina-3,7 (11) diene (SQT). The maize scent was found to be a mixture of three GLVs, six MTs, two homoterpenes (HTs), and 12 SQTs (Table 1a). The amounts of each component varied considerably throughout the 24-h cycle. The relative ratios of MT and SQT changed from day to night (Fig. 2). The peak SQT emission occurred during the day. In contrast, the peak MT emission was observed at night and dawn. HT was always present in low quantities, and the quantity did not change substantially over time. The diel GLV composition was characterized by the absence of Z3-6:Ac during the day and at dawn, but the relative amount of Z3-6:OH increased at dusk.
MeSA (an induced GLV) and !-copaene, were the two main compounds in maize headspace collections; they accounted for half of all the VOCs detected (Table 1a). The relative amounts of MeSA and !-copaene varied over time. The diel variations in these two VOCs were clearly separate at dusk and dawn. The ratio of MeSA to !-copaene in the headspace varied from 0.39 at dusk to 2.32 at dawn. The ratio between day and night was less impressive; it ranged from 1 during the day to 0.68 at night (Fig. 3). During the day, the emission rates of !-copaene and MeSA were nearly equal. At dusk, however, individual maize plants emitted about 2.5 times more !-copaene than MeSA. At night, they tended to be emitted at similar levels At dawn, the emission rates were the reverse of those at dusk, and maize plants emitted about 2.3 times more MeSA than !-copaene. The three PCs calculated from the measured amounts of VOCs to compare time periods explained 35, 21, and 14 % of the variance, respectively. A MANOVA performed on the three PC values revealed significant differences in the ratios of maize VOCs among the four time periods (Wilk’s lambda = 0.071, p = 0.0015). When the three PC’s were analysed separately, only the scores of PC2 differed significantly between time periods (two-way ANOVA, with date as an additional factor: F 3,11 = 15.7, p = 0.0002) and captured the time-related variance of the VOC ratio (Fig. 4). Pairwise comparisons of PC2 scores among the four periods showed no significant differences between the maize VOCs ratios for day and dusk, or night and dawn. All other comparisons showed a significant difference (Tukey test: p<0.02).
Separate analyses were performed on the headspace VOC data for different classes of compounds. We found that the SQT profiles differed among time periods (Wilk’s lambda = 0.060, p = 0.006). Again, the profiles for night and dawn, and for day and dusk were similar; but pairwise comparisons among other time points attained significance. In contrast, no among-period differences could be shown for the other compound classes (Wilk’s lambda = 0.27, p = 0.15); i.e., GLV, HT, MT.
The among-period differences for individual VOCs was analysed with the Kruskal-Wallis test (Table 1a). In the individual maize static headspace collections, the diel relative amounts of three VOCs out of 21 changed significantly (Z3-6:Ac [df=3, N=16, Z=8.45, p=0.038], trans-!-bergamotene [df=3, N=16, Z=10.25, p=0.017], and !-cadinene [df=3, N=16, Z=12.91, p=0.005]). Pairwise comparisons for the GLV, Z3-6:Ac, showed no significant changes in the relative ratio over time (p0.05). However, Z3-6:Ac was detected only at dusk and night in maize VOC collections. Trans-!-bergamotene and !-cadinene were not detected at night or dawn; they were present only during the photophase, with a significantly higher amount at dusk (p=0.038, Z=8; p=0.013, Z= 8.500 respectively).

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Maize field atmosphere VOCs

In the maize field atmosphere, a total of 13 VOCs were detected and identified (Table 1b). The VOCs profile was dominated by MeSA and a complex of p-cymene with limonene. There was also a constant low level of dimethyl nonatriene (DMNT). The ratio of MeSA to the complex of p-cymene-limonene did not change with diel periods; !-pinene, 3-carene, linalool, !-copaene, « -farnesene, and trans-nerolidol were detected in random amounts in the atmosphere without a clear diel pattern. None of the 13 VOCs detected in the maize field significantly changed in amount over time. Furthermore, when the 13 VOCs were grouped into chemical classes, no significant diel variation was observed for the relative amounts (Kruskal Wallis test, p0.05 for all cases).
There were 12 VOCs in common between samples from the maize field atmosphere and the maize headspace. Out of the 13 VOCs detected in the maize field atmosphere, the MT alcohol, linalool, although repeatedly detected in the atmosphere, was the only VOC that was not found in the maize headspace. The VOC profiles from maize headspaces were considerably richer than the profiles from the field air samples. The headspace maize VOC blend mainly comprised SQT; in contrast, the maize field atmosphere mainly comprised MT. In general, the diel pattern changes in the VOC composition for individual maize plants did not match the patterns observed in the maize field atmosphere. Only MeSA (GLV) had the same diel pattern of emission in the field and the headspace collections; the emission peaked while it was light over the 24-h cycle, and it decreased at dusk and night.

Insect race confirmation

The pheromone races of the female moths were confirmed by Solid Phase Micro Extraction (SPME; (Frérot et al. 1997) using carbowax/divinylbenzene fibres (65 $m, Supelco Inc.). The pheromone gland was extruded by gentle pressure on the abdomen and kept in this position with metallic forceps. A SPME fibre was gently rubbed on the gland and then either directly analyzed or wrapped in aluminum foil and stored at -20C until analysis. Pheromones were identified using a gas chromatograph (GC; Varian 3400Cx) equipped with a split-splitless injector and an Rtx ®-Wax column (Restek; 30 m x 0.32 mm i.d., film thickness 0.25 $m). The compounds absorbed on the fibre were thermally desorbed in the injector (splitless mode), which was maintained at 240 °C. The oven temperature was programmed from 50 °C to 100 °C at 15 °C/min; 100 °C to 245 °C at 5 °C/min; helium (15 psi) was the carrier gas. Pheromone compounds were identified by comparison of the retention times of the natural compounds with those of the synthetic reference samples. The ratios of the various compounds were calculated from the peak areas of the products.

Wind tunnel bioassays

The experiments were carried out in a half-cylinder wind tunnel (190 cm long % 80 cm wide % 45 cm high) with an airflow of 0.6 m/s at 23±2 °C and with ~60% relative humidity. A constant red incandescent light source above the tunnel allowed observations and video recording. Three-to-five-day-old mated females in individual capped wire mesh cages (3%6 cm) were released from a 12 cm-high platform 150 cm downwind of the source. Bioassays were carried out between the third and the sixth hour after the onset of scotophase. Plant odour came from potted maize, mugwort, or hop plants that were kept under the same photoperiod as the insects. Plants were placed in the upwind part of the tunnel. Pairwise oviposition choice-tests of maize vs. mugwort and maize vs. hop were performed in a randomized fashion. Each test lasted 15 min or ended earlier if the female began to oviposit. Flight behaviour was recorded
on a hard-drive (Archos AV500) with a COHU Solid State Camera equipped with an Avenir TV lens 4.8 mm F1.8 (Japan). We recorded the following behavioral steps of the female moths: activation, taking flight, upwind orientation, landing, and oviposition. Each female was tested once, and a total of 51 E-race moths and 49 Z-race moths were tested.

Table of contents :

List of tables
List of figures
List of original publications
Introduction
Study objectives
Insect-plant relationship
Chapter 1: Study subjects
European corn borer
Chemical communication in European corn borer
Taxonomy of European corn borer pherotypes
European corn borer in agriculture
European corn borer host plants
Maize
Mugwort
Hop
Chapter 2: Volatile organic compounds
Volatile organic compounds in plants
Green leaf volatiles
Terpenes
Monoterpenes
Sesquiterpenes
Homoterpenes
Inducible volatile organic compounds
Olfactory environment
Paper I Chemical landscape of maize field for host-seeking moth
Paper II Diel patterns of volatile organic compounds released by maize plants: The chemical environment of the Ostrinia nubilalis moth
Chapter 3: Host plant specialization
Host plant choice and recognition
European corn borer host plants
Paper III Volatile organic compounds and host-plant specialization in European corn borer E and Z pheromone races
Chapter 4: Assortative mating
Male scent organs
Male pheromones
Courtship behaviour
European corn borer courtship behaviour
Speciation
Sympatric speciation
Paper IV Male hairpencils and assortative mating in European corn borer pherotypes
Discussion and perspectives
References

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