Mature leaves of C. x deliciosa, Z. ailanthoides, Z. schinifolium., Z. piperitium, P. amurense, O. japonica, S. japonica, E. meliifolia, R. graveolens, F. vulgare, A. keiskei, and H. lanatum, were collected from plants cultured in the garden of Tsukuba, Ibaraki, Japan. C. deliciosa, R. graveolens, F. vulgare, and A. keiskei leaves were procured from local gardening stores, E. meliifolia was replanted from Ishigaki Island of the Nansei Islands, and other plants originated from the forest where the butterflies for our experiments were collected. Selection criteria of these plants included the following: the plant is (1) distributed in most areas of mainland Japan (except E. meliifolia, which occurs only in Kyushu, Shikoku, and south–end of Honshu), (2) abundant, and (3) the major hostplant of at least one Papilio species occurring in mainland Japan. Although R. graveolens and F. vulgare are alien species in Japan, the former is used by many Rutaceae–using Papilio species, whereas the latter is used extensively by P. machaon in most of the Northern Hemisphere.
Flowers from C. trichotomum (Lamiaceae), C. japonica (Vitaceae), R. pseudoacacia (Fabaceae), L. japonica (Caprifoliaceae), C. deliciosa, and Z. ailanthoides were collected from Tsukuba city in their respective flowering seasons. Identification of these plants were done by Inoue TA according to abundant illustration books published in Japan.
For the identification of volatile substances in the leaves and flowers, all plant specimens were collected around noon, when the production of plant volatiles is expected to be the maximal in the day. Samples were transferred to the Showa Denko Materials Techno Service laboratory and stored at -40 ºC until further analysis.
Analysis of plant volatiles (Method– 1)
The leaf volatiles were subjected to gas chromatography–mass spectrometry (GC–MS) on an Agilent system consisting of a model 7890 gas chromatograph, a model 5977 mass–selective detector (EIMS, electron energy of 70 eV), and an Agilent ChemStation data system (Santa Clara, CA, USA). Volatiles in the leaf were trapped into an odorant–collecting cartridge (Tenax TA, GL Sciences, Tokyo, Japan) and subjected to GC using a newly developed non–solvent method known as dynamic–headspace and thermal–desorption system (Gerstel, Überhausen, Germany) with COMPS2XLxt multi–purpose sampler. The GC column was a DB–VRX column (Agilent, USA) with a film thickness of 1.44 µm, length of 60 m, and internal diameter of 0.25 mm. The carrier gas was helium, with a flow rate of 2.1 mL/min. The GC oven temperature was regulated as follows: 40ºC initial temperature held for 3 min; increased at 5ºC/min to 260ºC and held for 8 min. Leaf samples (0.2 g) were placed into 20 mL vials and measured by the dynamic–headspace technique. Mass–selective detector was set at 230ºC. The volatiles were identified by comparing their MS fragmentation patterns to those in the MS library (NIST14 database).
Analysis of volatiles from A. keiskei (Method–2)
A. keiskei leaves were dried at 40 ºC, crushed to a powder, and the volatiles from 0.2 g of this powder were trapped at room temperature = 22°C for 24 h using an odorant–collecting cartridge (RCC18; GL Science). RCC18 was dipped in acetone (Wako, Fujifilm, Ôsaka, Japan) to extract volatiles that were then analyzed on a GC–MS QP2010 system (Shimadzu, Japan). The GC column was a DB–5MS column (Agilent, USA) with a film thickness of 0.25 µm, length of 30 m, and internal diameter of 0.25 mm. The carrier gas was helium. The GC oven temperature was regulated as follows: 40ºC initial temperature held for 2 min; increased at 5ºC/min to 240ºC and held for 5 min. Mass–selective detector temperature was set at 250ºC. The volatiles were identified by comparing their MS fragmentation patterns to those in the MS library (NIST14 database). This analysis was performed as bachelor thesis of Otani.