Table Of Content蝶と蛾 Lepidoptera Science 68（2）: 61-64, August 2017
Volatile compounds from a Parnassiinae butterfly, Luehdorfia japonica（ Lepidoptera, Papilionidae）
Kento Yoshimori1）, Taro Noguchi2）, Shinji Ohta2） and Hisashi Ômura2）
1） Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima, 739-8528 Japan
2） Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima, 739-8528 Japan
Abstract Male adults of several species in the family Papilionidae emit characteristic odors. However, it remains
unclear whether the presence of male-specific volatiles is a common trait in this family, because the chemistry of adult
odors has been investigated in only a limited number of species belonging to the Papilioninae subfamily. We
conducted gas chromatography–mass spectrometry analyses of the highly volatile substances of the Parnassiinae
butterfly, Luehdorfia japonica（ Lepidoptera, Papilionidae）, using solvent extracts of laboratory-reared adults.
Although this species lacks a strong odor detectable by the human nose, a total of 24 highly volatile compounds were
identified from both sexes, and 17 of these compounds were discovered in adult papilionid butterflies. The most
abundant component was limonene, which was present at a level of 1 (cid:9602)g per individual on average. Both sexes
showed similar compositions of these volatile compounds, with no significant differences in the quantity of each
compound between sexes. These results suggest that L. japonica adults possess a common odor in both sexes and
lack male-specific volatiles.
Key words GC–MS analysis, Luehdorfia japonica, scent substance, solvent extraction.
Introduction Stoll contain small amounts of linalool（ Ômura and Honda,
2005; Ômura et al., 2012）. However, whether the presence of
Adult lepidopteran insects produce sex-specific odors for mating;
male-specific volatiles is common in other subfamilies such as
female moths emit blends of volatile compounds that serve as
Baroniinae and Parnassiinae remains to be investigated. In this
sex pheromones, and male butterflies have characteristic scents
study, we examined the adult scent chemistry of a Parnassiinae
（Boppré, 1984; Vane-Wright and Boppré, 1993）. Several
butterfly, Luehdorfia japonica Leech using gas chromatography-
butterfly species whose males possess scents that are perceptible
mass spectrometry（ GC–MS）. L. japonica is a univoltine,
to the human nose have been subjected to chemical analyses, and
spring-flying species, endemic to Honshu, Japan. Its larvae feed
various compounds have been identified as male-specific
on leaves of wild ginger, particular species of the genus Asarum
volatiles（ Honda, 1986a,b; Ômura, 2015）. In several butterfly
（Aristolochiaceae）. Because the essential oils of their host
species, male-specific volatiles are produced by specialized alar
plants are abundant in odoriferous compounds, we also examined
organs（ androconia） and play important roles in mating
the relationship between the odor of the butterfly and the
（Kristensen and Simonsen, 2003）. However, information on
volatiles of its host plant.
scent chemistry is available for only a limited number of
butterfly species, unlike moths.
Materials and methods
In the family Papilionidae, chemical profiles of adult odors have
Insects
been determined only in species belonging to the subfamily
Papilioninae, in which particular terpenoids have been identified Luehdorfia japonica adults were raised from eggs in the
as male-specific volatiles. Byasa alcinous alcinous（ Klug） males laboratory. Two gravid females were collected in the field at
are characterized by a strong musk-like odor, which consists of Higashihiroshima（ 34.23(cid:155)N, 132.42(cid:155)E, Hiroshima Prefecture）
γ-himachalene acetate（ the predominant component） and and placed in plastic containers（ 35×50×25 cm） to allow them
several sesquiterpene hydrocarbons, while males of the to lay eggs on fresh leaves of Asarum hexalobum（ synonym
subspecies Byasa alcinous loochooana（ Rothschild） have an Heterotropa hexaloba） F. Maek. under incandescent lamps. The
odor with a slightly different composition（ Itoh and Ômura, larvae were reared on fresh leaves at 20(cid:155)C under a 16 L:8 D
2015; Ômura et al., 2016）. Male Papilio machaon hippocrates C. photoregime. The adults, provided with 15% aqueous sucrose
& R. Felder emit a fragrant smell containing linalool and geranyl once daily, were maintained individually in plastic cases（ 9 cm
acetone as the major substances（ Ômura et al., 2001）. The in height, 12 cm in inner diameter）.
scents of male Papilio polytes L. and Papilio protenor demetrius
62 K. Yoshimori et al.
Extraction nonanal, and decanal）, one aliphatic ketone（ 2,5-hexadione）,
two aliphatic esters（ isobutyl isobutyrate and ethyl 2-acetyl-3-
At 3 days after eclosion, adult butterflies were killed by freezing
methylbutyrate）, one aliphatic lactone（ γ-dodecalactone）, three
at –20(cid:155)C and subjected to 3 min extraction with purified
aromatic hydrocarbons（ o-, m-, and p-xylenes）, three
dichloromethane. Without dissection, individual adults（ 10
oxygenated aromatic compounds（ benzaldehyde, benzofuran,
males and 8 females） were extracted intact with 5 mL solvent at
and acetophenone）, and four monoterpenoids（ p-cymene,
room temperature. The crude extracts were subsequently filtered,
limonene, rose oxide, and pulegone） were commercial products
concentrated to 100 μL under a stream of nitrogen gas, and
from Tokyo Chemical Industry（ Tokyo, Japan） or Sigma-
stored at –20(cid:155)C until chemical analyses were conducted.
Aldrich（ St. Louis, MO）. Authentic longifolene, a component of
an essential oil from the Atlas cedar（ Cedrus atlantica）, was
Chemical analyses
supplied by Pranarôm International（ Ghislenghien, Belgium）.
The concentrated extracts were subjected to GC–MS using a
Shimadzu QP5000 mass spectrometer coupled with a Shimadzu Results
GC-17A gas chromatograph equipped with an Agilent J&W HP-
The total ion chromatograms of the male and female extracts
5MS capillary column（ 30 m×0.25 mm ID, 0.25 μm film
showed few large peaks within the retention time range of 4–20
thickness） at an electron ionization（ EI） voltage of 70 eV. All
min, indicating that adult L. japonica of both sexes have small
samples were injected in the splitless mode at 280(cid:155)C, using
amounts of highly volatile compounds（ Fig. 1A）. This result is
helium as the carrier gas. The oven temperature was
consistent with the observation that adults of this species lack
programmed from 40(cid:155)C（ 2 min isothermal） to 300(cid:155)C（ 10 min
strong odors detectable by the human nose. However, comparing
isothermal） at a rate of 10(cid:155)C/min. For three compounds, high-
the retention times and mass spectra with those of authentic
resolution gas chromatography-time-of-flight mass spectrometry
samples, we first identified 21 volatile compounds: four alkanes
（HR–GC–TOF–MS） was performed, using a Jeol JMS-
（peaks 8, 13, 18, and 21）, three aliphatic aldehydes（ 4, 14, and
T100GCV 4G mass spectrometer equipped with an Agilent J&W
19）, one aliphatic ketone（ 6）, two aliphatic esters（ 5 and 15）,
HP-5 capillary column（ 30 m×0.32 mm ID, 0.25 μm film
one aliphatic lactone（ 23）, three aromatic hydrocarbons（ 1–3）,
thickness） at an EI voltage of 70 eV or a chemical ionization
three oxygenated aromatics（ 7, 8, and 12）, and four
（CI） voltage of 200 eV（ with isobutane as the CI gas）.
monoterpenoids（ 9, 10, 16, and 20）（ Fig. 1B, Table 1）. Then
Compounds were identified by comparing retention times and
three other compounds（ 11, 17, and 22） were analyzed by high-
mass spectra with those of authentic samples. As authentic
resolution mass spectrometry.
samples for two compounds were unavailable, tentative
identification was performed using the NIST/EPA/NIH mass Peak 11 had a base ion at m/z 55（ 100%）, a possible molecular
spectral library version 2.0 g, which had been packaged with the ion at m/z 156（ 15%）, and fragment ions at m/z 141（ 11%）, 127
mass spectrometer. Quantitative estimates of individual （7%）, 111（ 26%）, 95（ 28%）, 83（ 59%）, 67（ 50%）, and 41
compounds were obtained by comparing peak intensities with （98%） in GC–MS. The molecular formula was established as
those of authentic linalool. CH O from the molecular ion [M]+ at m/z 156.11517 in HR–
9 16 2
GC–TOF–MS in EI mode（ calcd for CH O: 156.11503）. This
9 16 2
Authentic samples compound was tentatively identified as ethyl 3,4-dimethyl-2-
pentenoate.
The authentic samples of four alkanes（ decane, undecane,
dodecane, and tridecane）, three aliphatic aldehydes（ heptanal, Peak 17 showed a base ion at m/z 57（ 100%, relative ion
Fig. 1. Typical total ion chromatograms of the crude extracts of Luehdorfia japonica males（ upper） and females（ lower） in GC–MS.
（A） Total ion chromatograms for retention times from 4 to 38 min.（ B） Enlarged chromatograms for retention times from 4 to 20
min. Peak numbers correspond to those in Table 1.
Adult odor of Luehdorfia japonica butterfly 63
Table 1. Chemical composition of volatile compounds in the crude extract of Luehdorfia japonica adults
Retention Retention index in the Amount/individual（ ng, mean ± SD）
Peak No. Compound
time（ min） HP-5MS column Male（ n = 10） Female（ n = 8）
1 5.94 865 m-Xylene 758 ± 432 453 ± 124
2 6.09 873 p-Xylene 804 ± 458 497 ± 137
3 6.53 896 o-Xylene 322 ± 213 182 ± 79
4 6.71 905 Heptanal 89 ± 70 51 ± 24
5 6.98 920 Isobutyl isobutyrate 257 ± 160 122 ± 85
6 7.23 934 2,5-Hexadione 1268 ± 821 715 ± 411
7 7.85 967 Benzaldehyde 110 ± 67 68 ± 24
8 8.47 1000 Decane+Benzofuran 269 ± 161 168 ± 60
9 9.00 1030 p-Cymene 1061 ± 665 662 ± 191
10 9.08 1034 Limonene 1631 ± 979 1076 ± 405
11 9.26 1044 （Ethyl 3,4-dimethyl-2-pentenoate）a 961 ± 491 585 ± 200
12 9.77 1073 Acetophenone 141 ± 92 83 ± 37
13 10.26 1100 Undecane 652 ± 357 472 ± 197
14 10.36 1106 Nonanal 208 ± 114 124 ± 54
15 10.43 1111 Ethyl 2-acetyl-3-methylbutyrate 217 ± 119 106 ± 49
16 10.50 1115 Rose oxide 150 ± 95 104 ± 46
17 10.68 1125 （1,1-Dibutoxybutane） 1308 ± 733 793 ± 325
18 11.90 1200 Dodecane 249 ± 144 261 ± 108
19 12.03 1208 Decanal 97 ± 59 55 ± 21
20 12.67 1250 Pulegone 64 ± 37 47 ± 21
21 13.44 1300 Tridecane 68 ± 40 100 ± 54
22 15.27 1428 Longifolene 43 ± 18 46 ± 20
23 18.64 1691 γ-Dodecalactone 81 ± 67 69 ± 64
a Compounds in parentheses are tentatively identified
intensity） and fragment ions at m/z 43（ 14%）, 73（ 18%）, 103 abundant component, with an average of > 1μ g per individual in
（5%）, 129（ 7%）, and 159（ 2%）, without a clear molecular ion. both sexes, followed by 1,1-dibutoxybutane and 2,5-hexadione.
This fragmentation pattern suggested a possible aliphatic acetal The chemical composition of highly volatile compounds was
structure. However, we could not obtain [M]+ and [M+H]+ ions very similar between sexes, with no significant differences in the
of this compound in exact mass measurements. Following a quantity of each compound between the sexes（ p > 0.05, Mann–
mass spectral library search, 1,1-dibutoxybutane was tentatively Whitney U test）.
assigned to it as its structure.
Discussion
The EI–MS fragmentation pattern of peak 22 was typical of a
cyclic sesquiterpene, although a molecular ion peak was not Luehdorfia japonica adults, even males, had little odor detectable
observed at m/z 204. As HR–GC–TOF–MS in the CI mode by the human nose. However, crude extracts of it contained
showed a [M+H]+ ion at m/z 205.19617（ calcd for C15H25: various highly volatile substances, such as aliphatic, terpenoid,
205.19563）, its molecular formula was established as C15H24. and aromatic compounds, although in small amounts. Of the 24
This compound was identified as longifolene by comparing the compounds identified, heptanal, benzaldehyde, p-cymene,
EI–MS spectrum and retention index of the component in Atlas limonene, nonanal, dodecane, and decanal have previously been
cedar oil. reported as adult scent substances in the subfamily Papilioninae,
while the remaining 17 compounds were discovered in the
Of the 24 compounds identified, limonene was the most
64 K. Yoshimori et al.
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Kristensen, N.P. and T.J. Simonsen, 2003. ʻHairsʼ and cells. In
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Ômura, H., N. Yanai and K. Honda, 2012. Sexual dimorphisms in
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geographical variation in several traits, such as larval host usage himachalenes in male-specific odor of the Chinese windmill
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Acknowledgments
We thank Dr Tomoko Amimoto, the Natural Science Center for
摘 要
Basic Research and Development（ N-BARD）, Hiroshima
University, for the measurement of HR–GC–TOF–MS. ウスバアゲハ亜科ギフチョウLuehdorfia japonicaの成虫揮
発性成分（吉森健人・野口太郎・太田伸二・大村 尚）
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