Collection of halitosis from Japanese Apaturinae larvae
The experiments were conducted at Sasakia charonda research center, Kashihara, Nara. Sasakia charonda larvae were reared in a 6.5 m × 5 m × 3 m (L × W × H) cage covered with a 1-mm nylon mesh net, and fed C. sinensis fresh leaves. We also used S. charonda larvae reared in the “Museum of Sasakia charonda in Hokuto, Yamanashi”. The sources, dates of origin, and additional information on the butterflies used in these experiments are shown in Table 1. Halitosis of 4th–6th instar larvae of S. charonda, H. persimilis, and A. metis were collected in sampling tubes (Tenax TA, GL Sciences, Japan) using an air suction pump with an initial suction capacity of approximately 20 L/min. To induce larval mouth opening, we pinched the posterior abdominal segments of the larvae with thumb and index finger. Upon this stimulation, larvae raised their heads and opened their mandibles, and the halitosis were collected (Fig 1). Because the amounts of each chemical were thought to be very small, we combined the halitosis expelled by 10 individual larvae of one species into each sampling tube. For collection of volatiles from H. assimilis larvae, a manual pump was used. The collection time for each larva was 15 s. Air just above the host-plant leaf surface was used as control. GC–MS analysis was performed on the samples at the Analyzing Center, Showa Denko Materials Techno Service.
Analysis of volatiles
The halitosis compounds were subjected to GC–MS analysis on an Agilent system consisting of a model 7890B gas chromatograph, a model 5977A mass-selective detector (EIMS, electron energy of 70 eV), and an Agilent ChemStation data system (Santa Clara, CA, USA). The GC column was a DB-VRX column (Agilent, USA) with 1.40 μm film thickness, 60 m length, and 0.25 mm internal diameter. The carrier gas was He, with a flow rate of 2.1 mL/min. The collected volatiles were analyzed using the thermal desorption method. The GC oven temperature program was 40 °C initial temperature for 3 min, followed by a temperature increase of 5 °C/min to 260 °C, which was held for 8 min. The mass-selective detector temperature was set to 230 °C. Sample components were identified by comparison of their mass spectral-fragmentation patterns against those stored in the MS library (NIST14 database) and by comparison to standards.
Reagents
As the results, we could detect 22 substances in the halitosis of S. charonda larvae. From these, we purchased 13 reagents, 2,3-butanedione (Wako, Fujifilm, purity > 98 %), 2-butanol (Wako, Fujifilm, purity > 99 %), 2-methyl-3-buten-2-ol (Tokyo Chemical Industry Co Ltd, purity > 97.0 %), 1-penten-3-ol (Wako, Fujifilm, purity = 95 %), 3-pentanol (Tokyo Chemical Industry Co Ltd, purity > 98 %), 3-pentanone (Wako, Fujifilm, purity = 98.0+%), 3-hydroxy-2-butanone (Tokyo Chemical Industry Co Ltd, purity > 98.0 %, may exist as crystalline dimer), 3-methyl-3-buten-1-ol (Tokyo Chemical Industry Co Ltd, purity >98 %), (±)-2-methyl-1-butanol (Wako, Fujifilm, purity ?), 2,3-butandiol (Tokyo Chemical Industry Co Ltd, purity > 97.0 %), 3-methyl-2-butenal (Tokyo Chemical Industry Co Ltd, purity > 97.0 %), 3-hexen-1-ol (Wako, Fujifilm, purity ≥ 95 %), 2-methyl-2-pentenal (Tokyo Chemical Industry Co Ltd, purity > 97.0 %), and Cyclohexanol (Wako, Fujifilm, purity = 98 %), which we could obtain easily. These were used to confirm substances we could detected and behavioral testing of ants and larvae described below.
We also purchased Prenol (Tokyo Chemical Industry Co Ltd, purity > 98.0 %), Hexanal (Wako, Fujifilm, purity = 95.0+%), Benzaldehyde (Wako, Fujifilm, 98.0+%), 6-methyl-5-hepten-2-on (Wako, Fujifilm, purity = 96.0+%), Nonanal (Wako, Fujifilm, purity = 95.0+%), and Geranylaceton (cis-trans mixture, Tokyo Chemical Industry Co Ltd, purity > 96.0%GC) detected from other three species performed same examination.
Calculation of Retention Index of detected substances
We could not find the Retention Index (RI) for DB-RVX, so we calculated RIs of the substances we could detected. First, we analyzed mixture of Pentane, Heptane, and “analytical standard, contains C8-C20, ~40 mg/L each, in hexane, MERCK”, to know the retention times of those standard substances. Next, mixed reagents we could purchase, analyzed in the same manner as the halitosis samples, and the RI of these reagents were calculated. The RI of other substances were estimated from the RI of the substances before and after those.
For this calculation, we used the equation shown below.
IA = 100 * n + 100 * (logtA – logtn) / (logtn+1 – logtn)
Here, n are carbon number of n- H-(CH2)n-H, tn are retention time of n-alkane, and tA is the retention time of the substance that we want to calculate retention index those each retention time are located between those of tn+1 and tn.
Effect of S. charonda halitosis on predator ants
This experiment was performed in the Sasakia charonda research center, Kashihara, Nara and Museum of Sasakia charonda in Hokuto, Yamanashi. Twelve confirmed substances were used to investigate their effects on Pristomyrmex punctatus and Formica japonica ants. We soaked a cotton swab with each substance and placed the swabs individually near the ants (Fig 2), and we observed whether the ants showed behavior to escape from the swabs soaked with the sample. The behavioral responses were recorded by a video camera (Standard resolution NTSC video, 720 × 480, HDR-HC7, SONY, Japan).
Effect of the S. charonda halitosis on the four Apaturinae species larvae
The substances identified in the rearing houses in Kashihara and Hokuto were presented to S. charonda larvae using an olfactometer. One side of a silicone tube was connected to the outlet of an air pump, and the other side of the tube was connected to polyethylene pipettes. Small pieces of filter paper soaked in each volatile were inserted into each pipette as the source of substance (Fig. 3A). For the other three species, substance presentation was performed with swabs in the same manner as for the ants (Fig. 3B). We changed the substances by exchanging pipettes or swabs. We observed the behavior of the larvae in response to these substances.