Sex pheromones of two Melittini species, Macroscelesia Japona and M. Longipes: Identification and field attraction

Article abstract of DOI:10.1007/s10886-006-9242-5

Two Melittini species, Macroscelesia japona and M. longipes (Lepidoptera: Sesiidae), are native to Japan, but occupy different localities as their host plants seldom grow together. The contents of the sex pheromone gland of adult females of both species, obtained after rearing larvae collected from the field, were investigated by gas chromatograph-electroantennogram detection (GC-EAD) and gas chromatograph-mass spectrometry (GC-MS) analyses. Two GC-EAD-active components were found in a crude extract of M. japona female pheromone gland, and identified as (2E,13Z)-2,13-octadecadien-1-ol (E2,Z13-18:OH) and (2E,13Z)-2,13-octadecadienal (E2,Z13-18:Ald). The average ratio of these two components was about 1:10. In the field, M. japona males were attracted to traps baited with E2,Z13-18:Ald alone, but the strongest attraction was observed with a 1:100 mixture of E2,Z13-18:OH and E2,Z13-18:Ald. The same two components were found in extracts of M. longipes females, but in a markedly different ratio. Male M. longipes were attracted most strongly to lures containing a 20:1 mixture of E2,Z13-18:OH and E2,Z13-18:Ald, although some males were also attracted to lures with E2,Z13-18:OH alone. Although the two species do not generally occur in sympatry, our data indicate that, in the event of overlap, cross attraction of the two species is unlikely.

Full text of DOI:10.1007/s10886-006-9242-5

J Chem Ecol (2007) 33:591–601
DOI 10.1007/s10886-006-9242-5
Sex Pheromones of Two Melittini Species, Macroscelesia
Japona and M. Longipes: Identification
and Field Attraction
Hideshi Naka & Shin-Ichi Inomata & Kanae Matsuoka &
Masanobu Yamamoto & Hajime Sugie & Koji Tsuchida &
Yutaka Arita & Tetsu Ando
Received: 20 August 2006 /Revised: 30 October 2006 /Accepted: 6 November 2006 /
Published online: 31 January 2007
#
Springer Science + Business Media, LLC 2007
Abstract Two Melittini species, Macroscelesia japona and M. longipes (Lepidoptera:
Sesiidae), are native to Japan, but occupy different localities as their host plants seldom
grow together. The contents of the sex pheromone gland of adult females of both species,
obtained after rearing larvae collected from the field, were investigated by gas
chromatograph-electroantennogram detection (GC-EAD) and gas chromatograph-mass
spectrometry (GC-MS) analyses. Two GC-EAD-active components were found in a crude
extract of M. japona female pheromone gland, and identified as (2E,13Z)-2,13-
octadecadien-1-ol (E2,Z13-18:OH) and (2E,13Z)-2,13-octadecadienal (E2,Z13-18:Ald).
The average ratio of these two components was about 1:10. In the field, M. japona males
were attracted to traps baited with E2,Z13-18:Ald alone, but the strongest attraction was
observed with a 1:100 mixture of E2,Z13-18:OH and E2,Z13-18:Ald. The same two
components were found in extracts of M. longipes females, but in a markedly different
ratio. Male M. longipes were attracted most strongly to lures containing a 20:1 mixture of
:
H. Naka (*) H. Sugie
Entomology Group, Department of Biological Safety, National Institute for Agro-Environmental
Sciences (NIAES), Tsukuba, Ibaraki 305-8604, Japan
e-mail: chun@wakaba.com
:
:
:
S. Inomata K. Matsuoka M. Yamamoto T. Ando
Graduate School of Bio-Applications and Systems Engineering (BASE), Tokyo University
of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
K. Tsuchida
Laboratory of Entomology, Faculty of Applied Biological Science, Gifu University,
Gifu 501-1193, Japan
Y. Arita
Zoological Laboratory, Faculty of Agriculture, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan
Present address:
H. Naka
Lab. Coevolutionary interaction between insects and plants, JT Biohistory Research Hall,
1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan

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E2,Z13-18:OH and E2,Z13-18:Ald, although some males were also attracted to lures with
E2,Z13-18:OH alone. Although the two species do not generally occur in sympatry, our
data indicate that, in the event of overlap, cross attraction of the two species is unlikely.
.
.
Keywords (2E,13Z)-2,13-octadecadien-1-ol (2E,13Z)-2,13-octadecadienal
.
.
.
Clearwing moth Melittini Reproductive isolation Lepidopteran pheromones
Introduction
The Sesiidae is a family that includes over 1,000 diurnal species worldwide (Pühringer and
Kallies, 2004). Adults of most species have wasp-mimic stripes (yellow or red bands with a
black body), in a species-specific pattern that may be involved in mate recognition. In
addition to visual cues, sesiids use female sex pheromones in mating (Bûda and Karalius,
1985; Karalius and Bûda, 1993). Since the original study on Synanthedon pictipes and S.
exitiosa by Tumlinson et al. (1974), the sex pheromones of 15 species of Sesiidae have
been identified (Ando, 2006). These identifications, however, have been mainly conducted
in Europe and the United States, and information about Asian species is limited. About
30 yr ago, sex attractants for two agricultural pests, S. hector and S. tenuis, were discovered
by a field screening test in Japan by using a synthetic pheromone of S. pictipes and related
compounds (Yaginuma et al., 1976; Tamaki et al., 1977). However, no actual pheromone
identifications were published on the Japanese species in this family until our recent study
on Nokona pernix (Naka et al., 2006).
The Sesiidae are divided into two subfamilies, Sesiinae and Tintiinae, with the former
subdivided into six tribes: Synanthedonini, Paranthrenini, Melittini, Sesiini, Osminini, and
Cissuvorini. Pheromone studies worldwide have been carried out mainly with species in the
tribes Synanthedonini and Paranthrenini. Species in each of the six tribes occur in Asia, but
no species in the tribe Osminini are found in Japan (Arita and Ikeda, 2000; Pühringer and
Kallies, 2004). The tribe Melittini is one of the largest tribes of Sesiinae, comprising six
genera and 150 species in Asia, Africa, and the New World (Pühringer and Kallies, 2004).
However, pheromones have been reported only for one species, M. cucurbitae (Klun et al.,
1990). In Japan, six species in the tribe Melittini, two Macroscelesia species and four
Melittia species, are known (Arita and Ikeda, 2000). Both Macroscelesia species are
monophagous, feeding on different host plants in different habitats. Larvae of M. japona
burrow into the stems of Gynostemma pentaphyllum, whereas larvae of M. longipes bore
into the stems of Actinostemma lobatum (Arita and Ikeda, 2000). G. pentaphyllum grows
commonly in Japanese coppices, whereas A. lobatum is an endangered species, growing in
riverbeds. In Japan, almost all rivers are now enclosed between protective walls, virtually
removing all habitats for A. lobatum. M. longipes, which feeds exclusively on A. lobatum,
and has shown a consequent reduction in numbers.
Although G. pentaphyllum and A. lobatum seldom grow together, it is possible that
adults of the two moths might coincide as they have similar flight periods and their
distributions may be in close proximity. With this in mind, we set about to determine
whether the two closely related clearwing moths have different pheromone systems, as well
as to increase the information on pheromone components used by Asian sessiid species.
This report deals with the identification and field evaluation of the first pheromones from
clearwing moths in the tribes Melittini.

J Chem Ecol (2007) 33:591–601
593
Methods and Materials
Insects and Pheromone Extraction About 40 overwintering larvae of M. japona were
collected in Maihara City, Shiga Prefecture, in November 2003 and vernalized in a
refrigerator at 8 1°C without light for 2 mo. After vernalization, insects were kept in a
plastic cage at 25 1°C under a 15L: 9D photoperiod until they emerged as adults. Adults
were sexed based on their antennae (Arita and Ikeda, 2000). Under laboratory conditions,
females exhibited the calling position irregularly during the photophase. Therefore, we
observed the behavior of 1- or 2-d-old virgin females and excised their abdominal tips when
a calling position was observed. Abdominal tips were placed individually in hexane
(250 μl/female) for 5 min to extract the pheromone, before analysis by gas chromatog-
raphy- electroantennographic detection (GC-EAD; Struble and Arn, 1984) and gas
chromatography-mass spectrometry (GC-MS).
For the study on M. longipes, about 20 galls containing larvae were collected from a
riverbed near Lake Sanaru in Hamamatsu City, Shizuoka Prefecture, in August 2003. The
galls were kept at 25 1°C under a 15L-9D photoperiod until the adults emerged after 15–
20 days. As M. longipes females showed a calling position 1–4 hr after the beginning of the
photophase, their abdominal tips were excised 4 hr after the beginning of the photophase.
Extracts were made and analyzed as for M. japona.
Chromatography and Derivatization of the M. japona Pheromone The crude extract of
pheromone glands [10 female equivalents (FE)] was adsorbed on a microcolumn packed
with Florisil® (100–200 mesh, Floridin Co., 200 mg). The column was successively eluted
with 1 ml each of hexane and 5, 10, 20, and 50% diethyl ether in hexane. Each fraction was
concentrated by a gentle nitrogen stream and an aliquot (1 FE) analyzed by GC-EAD. The
5% diethyl ether fraction (9 FE), which included an electroantennogram (EAG)-active
aldehyde component, was treated with NaBH4 (0.5 mg) in 2-propanol (0.1 ml) for 3 hr at
room temperature. After mixing with water (1 ml), the crude products were extracted with
hexane (0.5 ml) and analyzed by GC-MS.
Chemicals (2E,13Z)-2,13-octadecadienyl acetate (E2,Z13-18:OAc) was supplied by Shin-
Etsu Chemical Co., Ltd. (Tokyo, Japan). The sample included traces (<2%) of some
unknown impurities, but not the corresponding alcohol. (2E,13Z)-2,13-octadecadien-1-ol
(E2,Z13-18:OH) was prepared by the hydrolysis of E2,Z13-18:OAc with methanolic
NaOH. Other geometric isomers of (2,13)-octadecadien-1-ol were synthesized from 1,9-
nonanediol. Their synthesis and structure confirmation by NMR analysis have been
reported in a previous paper (Naka et al., 2006). (2E,13Z)-2,13-octadecadienal (E2,Z13-18:
Ald), was obtained by oxidation of E2,Z13-18:OH with pyridinium chlorochromate in
CH2Cl2.1H NMR: δ (ppm) 0.90 (t, 3H, J=7.3 Hz), 1.2–1.4 (broad s, 16H), 1.50 (m, 2H),
2.02 (m, 4H), 2.33 (dt, 2H, J=7 and 7 Hz), 5.35 (m, 2H), 6.12 (dd, 1H, J=7.9 and 15.6 Hz),
6.85 (dt, 1H, J=6.8, and 15.6 Hz), 9.50 (d, 1H, J=7.9 Hz). 13C NMR: δ (ppm) 14.0, 22.4,
26.9, 27.2, 27.9, 29.2, 29.3, 29.4, 29.5 (× 2), 29.8, 32.0, 32.8, 129.8, 129.9, 133.0, 159.1,
194.2.
Analytical Instruments The GC-EAD apparatus and conditions have been reported (Naka
et al., 2006). Briefly, an HP-5890 Series II gas chromatograph, (Hewlett-Packard,
Wilmington, DE, USA) with a DB-23 capillary column (0.25 mm×30 m, J&W Scientific,
Folsom, CA, USA) was used for the analyses. The oven temperature was maintained at

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J Chem Ecol (2007) 33:591–601
100°C for 2 min, programmed at a rate of 20°C/min to 175°C, then at 6°C/min to 220°C,
and held at this temperature for 15 min. The effluent from the column was split at a ratio
of 1:1 into two lines, one of which led to a flame ionization detector (FID) and the other
to the EAD. GC-MS analyses were conducted in electron impact ionization mode (EI,
70 eV) using an HP-5973 mass spectrometer interfaced to an HP-6890 gas chromato-
graph, using the same column and conditions as for the GC-EAD analyses. The ion
1
source temperature was 230°C. H and 13C NMR spectra were recorded using a JEOL
Alpha 500 Fourier transform spectrometer at 500.2 and 125.7 MHz, respectively, for
CDCl3 solutions containing TMS as an internal standard.
Field Tests Attraction of M. japona males to synthetic lures was examined at coppices in
Ibaraki Prefecture. Test I was conducted in Chikusei and Tsukuba cities from June 30 to
July 12, 2003; test II was conducted in Tsukuba City from June 25 to July 10, 2003; and
tests III and IV were performed in Tsukuba City from June 17 to July 16, 2004. Test
chemicals were absorbed onto a white rubber septum (Sigma-Aldrich Co., St. Louis, MO,
USA), which was placed in the center of a sticky board trap (Takeda Chem. Co. Ltd.,
Tokyo, Japan). Traps were placed 1.5 m above the ground and at least 10 m from each
other. Numbers of captured males in each trap were counted every few days. After
counting, the traps were rotated to eliminate any positional effect (Latin square design;
Perry et al., 1980).
Trials testing M. longipes synthetic pheromone were conducted over 4 d in mid-August,
2004 at the riverbed of an oxbow in Fujimi City, Saitama Prefecture. As M. longipes is an
endangered species, we did not capture males with a sticky trap but recorded their behavior
around rubber septa baited with synthetic chemicals. Each septum was fixed to a riverside
plant with a safety pin, 50 cm above the ground.
Statistical Analyses The EAG data that used 1 ng of synthetic pheromone compounds (see
Fig. 3a and c) were analyzed by one-way ANOVA. Means that were significantly different
were separated by Tukey–Kramer’s HSD test (Sokal and Rohlf, 1995). For the EAG-dose
data, using synthetic E2,Z13-18 compounds and for the field trials, the data were log-
transformed before analysis by one-way ANOVA. Means that were significantly different were
separated by Tukey–Kramer’s HSD test. The level of significance in all tests was set at 5%.
Results
Identification of Pheromone Components of M. japona Male M. japona antennae were
stimulated by two components, I and II (Fig. 1a). Component I eluted from a Florisil®
column with 10% ether in hexane, indicating an alcohol, whereas component II eluted with
5% ether in hexane, indicating an acetate or aldehyde. The mass spectrum of component I
(Fig. 1b, c) showed M+ at m/z 266 (relative intensity: 1.4%), [M-H2O]+ at m/z 248 (10.1%),
and a series of fragment ions derived from straight-chain compounds, indicating
octadecadien-1-ol. The spectrum is similar to that of synthetic E2,Z13-18:OH, which is
easily distinguished from spectra of 3,13-octadecadien-1-ol by the relative intensity of M+
and [M-H2O]+ (Naka et al., 2006). The retention time (Rt) of Component I was the same as
that of authentic E2,Z13-18:OH, but different from those of the three other geometric
isomers [(2E,13E)-isomer, Rt 11.30 min; (2Z,13E)-isomer, Rt 11.46 min; (2Z,13Z)-isomer,

J Chem Ecol (2007) 33:591–601
595
Fig. 1 Analysis of the sex pheromone components of Macroscelesia japona by GC-EAD and GC-MS. (a)
GC-EAD analysis of pheromone extract (0.5 FE). (b) GC-MS analysis of extract (0.5 FE); total ion
chromatogram (TIC) and mass chromatograms monitoring diagnostic ions at m/z 266 (M+ of E2,Z13-18:
OH), m/z 248 ([M-18]+ of alcohol and [M-60]+ of acetate), 219, 205, and 191 of E2,Z13-18:OH and E2,Z13-
18:OAc, and m/z 264 (M+), 246 ([M-18]+), 235, 221, and 207 of E2,Z13-18:Ald. E2,Z13-18:OAc was not
detected in the crude extract. (c) Mass spectrum of Component I (E2,Z13-18:OH). (d) Mass spectrum of
Component II (E2,Z13-18:Ald)
Rt 11.67 min]. The mass spectrum of component II showed M+ at m/z 264 (4.4%) and [M-
H2O]+ at m/z 246 (4.4%), indicating an octadecadienal (Fig. 1c, d). This spectrum
corresponded closely to that of synthetic E2,Z13-18:Ald. Furthermore, NaBH4 reduction of
the 5% ether fraction produced E2,Z13-18:OH, indicating that Component II was E2,Z13-
18:Ald. Ten GC-MS injections (1 FE each) of different extracts allowed determination of
the mean amounts of the two components: 0.3 1.2 ng/female for E2,Z13-18:OH and 4.2
5.4 ng/female for E2,Z13-18:Ald. The two components were in a ratio of roughly 1:10,
although there was considerable variation among samples. No other candidate pheromone
chemicals, such as E2,Z13-18:OAc, could be detected (Fig. 1b).
Identification of Pheromone Components of M. longipes Antennae of M. longipes males
exhibited consistent EAG responses to two compounds with the same retention times as
components I and II of M. japona (Fig. 2a). GC-MS analysis of the pheromone extract
(Fig. 2a–d) confirmed that extract of M. longipes pheromone gland contained E2,Z13-18:
OH and E2,Z13-18:Ald. Three GC-MS injections (1 FE each) of M. longipes extract gave a
mean titer of E2,Z13-18:OH of 16.8 8.0 ng/female. E2,Z13-18:Ald was detected in one
sample (0.4 ng/female), but not in the other two.
EAG Activities of Synthetic Compounds EAG responses of male antennae to synthetic
compounds known to be pheromone components of Sesiidae were determined for the two

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J Chem Ecol (2007) 33:591–601
Fig. 2 Analysis of the sex pheromone components of Macroscelesia longipes by GC-EAD and GC-MS. (a)
GC-EAD analysis of pheromone extract (0.5 FE). (b) GC-MS analysis of extract (0.5 FE); total ion
chromatogram (TIC) and mass chromatograms monitoring diagnostic ions at m/z 266 (M+ of E2,Z13-18:
OH), m/z 248 ([M-18]+ of alcohol and [M-60]+ of acetate), 219, 205, and 191 of E2,Z13-18:OH and E2,Z13-
18:OAc, and m/z 264 (M+), 246 ([M-18]+), 235, 221, and 207 of E2,Z13-18:Ald. E2,Z13-18:OAc was not
detected in the crude extract. (c) Mass spectrum of Component I (E2,Z13-18:OH). (d) Mass spectrum of
Component II (E2,Z13-18:Ald)
Macroscelesia species (Fig. 3). M. japona antennae showed the greatest response to E2,
Z13-18:Ald, the more abundant component in the pheromone gland of females, and a
moderate response to E2,Z13-18:OH, the less abundant compound (Fig. 3a). Responses to
both compounds were greater than responses to all other compounds tested (ANOVA, df=6,
F=21.50, P<0.01). There were no significant differences in response of antennae with
respect to dose of three 2,13-dienyl compounds. However, the relative response to the three
different compounds was the same at all doses (E2Z13-18:Ald> E2Z13-18:OH> E2Z13-18:
OAc) (Fig. 3b). M. longipes antennae showed the greatest EAG response (greater than to all
other compounds tested; ANOVA, df=6, F=9.90, P<0.01) to E2,Z13-18:OH, the most
abundant compound in the female pheromone gland extract (Fig. 3c). Again, there were no
significant differences in response to antennae with respect to dose of the three 2,13-dienyl
compounds. However, EAG responses to E2,Z13-18:OH were greater than responses to the
other two compounds at all three doses tested (Fig. 3d).
Field Attraction of M. japona Males Of the single-component lures baited with one of the
2,13-dienyl compounds, only traps baited with E2,Z13-18:Ald caught male M. japona (Test
I, Table 1). When different amounts of E2,Z13-18:OH or E2,Z13-18:OAc were added to
E2,Z13-18:Ald, surprisingly, only traps baited with both E2,Z13-18:OAc and E2,Z13-18:
Ald caught males (Test II, Table 1). As treatments with E2,Z13-18:OAc appeared to shut
down trap capture to all other blends (including to that of E2,Z13-18:Ald alone), further
tests were conducted in which the effect of the alcohol component was evaluated in a field

J Chem Ecol (2007) 33:591–601
597
a M. japona
b M. japona
300
300
250
200
250
200
150
100
50
E2,Z13-18:OAc
E2,Z13-18:OH
E2,Z13-18:Ald
a
150
a
a
b
ab
100
a
b
c
b
c
ab
b
c
a
b
b
c
50
c
c
0
0
0
10-2
10-1
1
10
Dose (ng)
E2,Z13-18
E3,Z13-18 Z3,Z13-18
Compound
c M. longipes
d M. longipes
300
300
250
a
250
200
150
100
50
a
E2,Z13-18:OAc
E2,Z13-18:OH
E2,Z13-18:Ald
a
200
150
100
50
b
b
b
b
a
b
b
b
b
b
b
0
b
b
0
0
10-2
10-1
Dose (ng)
1
10
E2,Z13-18
E3,Z13-18 Z3,Z13-18
Compound
Fig. 3 EAG responses of Macroscelesia japona and M. longipes males. (a) EAG responses of antennae of
M. japona males to synthetic standards (1 ng). (b) Responses of antennae of the M. japona males to various
doses of (2E,13Z)-2,13-octadecadienyl compounds. (c) EAG responses of antennae of M. longipes males to
synthetic standards (1 ng). (d) Responses of antennae of the M. longipes males to various doses of (2E,13Z)-
2,13-octadecadienyl compounds. Responses are the means of at least ten antennae of M. japona and four
antennae of M. longipes. Values followed by a different letter are significantly different at P<0.05 (Tukey–
Kramer’s HSD test) in (a)–(c). In (d), values followed by a different letter are significantly different at P<
0.05 (Tukey–Kramer’s HSD test) for a given dose of the different compounds
about 4 km from the field testing mixtures of the aldehyde and acetate. Traps baited with
lures containing both E2,Z13-18:Ald and E2,Z13-18:OH caught significant numbers of
males (Test III, Table 1), with the greatest numbers (significantly greater than for all other
treatments) being caught in the 1:100 (E2,Z13-18:OH: E2,Z13-18:Ald) blend. Again binary

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J Chem Ecol (2007) 33:591–601
Table 1 Attraction of M. japona males to lures baited with synthetic (2E,13Z)-2,13-octadecadienyl
compoundsa
Content of C18 compounds (μg)
Mean number of catches (Mean SD)a
E2,Z13-18:OH E2,Z13-18:Ald E2,Z13-18:OAc
Test I:
0
1,000
0
0
0
100
300
0
0
0
0
100
50
100
300
500
0
1,000
0
0
0
21.7 4.0
0
0
0
0
0
0
0
0
1,000
0
0
0
0
Test II:
1,000
1,000
1,000
1,000
1,000
0
1,000
1,000
1,000
1,000
1,000
1,000
0
1,000
1,000
1,000
1,000
1,000
1,000
0
100 15.8 4.1a
300 10.5 6.4a
0
0
0
0
0
0
0
0
0
0
Test III:
1.0 1.0b
13.0 7.0a
2.3 3.2b
0.3 0.6b
0
0
0
Test IV:
0
0
0
0
0
0
0
0.7 1.2b
10 2.7 3.8a b
50 5.0 3.6a b
100 6.0 4.4a b
300 8.0 3.6a b
500 11.0 2.6a
0
0
a Test I was performed in two fields (N=3); Chikusei City (a private coppice under Kokai-Ohashi Bridge)
and Tsukuba City (coppices in NIAES) from June 30 to July 12, 2003. Test II was performed in Tsukuba
City (N=4); coppices in NIAES, NIES and Yukari-no-Mori from June 25 to July 10, 2003. Tests III and IV
were performed in the same fields as Test II (N=3) from June 17 to July 16, 2004.
b Values followed by a different letter are significantly different at the 5% level by Tukey-Kramer test.
blends of E2,Z13-18:Ald and E2,Z13-18:OAc caught significant numbers of male M.
japona (Test IV, Table 1).
Field Attraction of M. longipes Males Because M. longipes males fly slowly, their behavior
around synthetic lures could readily be observed in the field. Two behaviors were observed:
approaching the lure and touching the lure with forelegs. Males only approached lures
containing E2,Z13-18:OH (Table 2). About half of the approaching males touched the lures.
The greatest numbers of males that approached lures did so to binary blends of E2,Z13-18:
OH and E2,Z13-18:Ald; the numbers that approached the 1,000:50 and 1,000:300 lures
were significantly greater than the number that approached the lure containing only E2,
Z13-18:OH. Greater numbers of males touched the 1,000:50 lure (E2,Z13-18:OH: E2,Z13-
18:Ald) than touched any of the other lures. The numbers of males that approached or
touched the binary blend of E2,Z13-18:OH and E2,Z13-18:OAc were not significantly

J Chem Ecol (2007) 33:591–601
599
Table 2 Attraction of M. longipes males to lures containing synthetic (2E,13Z)-2,13-octadecadienyl
compoundsa
Content of C18 compounds (μg)
Mean number of attracted males (Mean SD)a
E2,Z13-18:OH
E2,Z13-18:Ald
E2,Z13-18:OAc
Touched
Attracted
1,000
1,000
1,000
1,000
1,000
0
0
50
100
300
0
0
0
0
0
100
0
6.3 1.7b c
13.8 3.3a
13.0 6.1a b
13.3 2.5a
5.5 2.4c
0
3.0 1.8b c
9.8 2.2a
5.3 2.9b
5.5 1.3b
3.0 1.4b c
0
1,000
0
0
0
1,000
0
0
0
0
0
0
a Test was performed in a riverbed of the Bin-Numa oxbow (Fujimi City, Saitama Prefecture) for 4 d between
August 12 and August 23, 2004.
b Mean number of 4 days. Values followed by a different letter are significantly different at the 5% level by
Tukey–Kramer test.
c Attracted: males attracted to within 5 cm. Touched: males touched the lure with their forelegs.
different from the numbers that approached or touched the lure containing only E2,Z13-18:
OH (Table 2).
Discussion
GC-EAD, GC-MS analyses of pheromone extracts, combined with field studies, revealed
E2,Z13-18:OH and E2,Z13-18:Ald as common pheromone components of two Macro-
scelesia species, M. japona, and M. longipes. The major component of the former species
was aldehyde and that of the latter was alcohol. These compounds were identified by
comparison with chemical data of known compounds, not by direct chemical reaction,
because of the limited number of available insects. All the dienyl compounds known as
Sesiidae pheromones or attractants have a C18-chain skeleton unsaturated at the 3- and 13-
positions or the 2- and 13-positions (Ando, 2006). The GC-MS data of the alcohol
component of both Macroscelesia species strongly indicated a 2,13-dienyl structure (Naka
et al., 2006). Configurations of the two double bonds were determined by comparing Rt
values of the natural component and the four geometric isomers. The second component
was indicated as an aldehyde by its elution time on the DB-23 column. The double-bond
positions and configurations of the aldehyde component from M. japona females were
determined by reduction by NaBH4 to produce the same dienyl alcohol as the other
pheromone component. To assist in pheromone identifications of other sessiid moths, we
intend to synthesize the four geometric isomers of 2,13-18:Ald.
Compounds with the C18 2,13-dienyl skeleton have been reported as sex pheromones in
several species in the lepidopteran families of Sesiidae, Cossidae, and Tineidae. All these
compounds have a (2E,13Z)-configuration; no 2,13-dienes with other configurations have
been found in pheromone gland extracts of female moths. (Ando, 2006; El-Sayed, 2006). In
the Sesiidae, E2,Z13-18:OH and E2,Z13-18:Ald have been identified only from Melittia
cucurbitae (Klun et al., 1990) and Sesia apiformis (Francke et al., 2004), respectively,

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J Chem Ecol (2007) 33:591–601
whereas E2,Z13-18:OAc has been identified in a number of species. Interestingly, Melittia
cucurbitae is taxonomically close to Macroscelesia spp.
The reproductive isolation of sibling species, through use of different blends of common
sex pheromone components, has been reported in many species of moths (Löfstedt et al.,
1991; Ishikawa et al., 1999). Our study revealed that two Macroscelesia species produce
the same two pheromone components, E2,Z13-18:OH and E2,Z13-18:Ald, but apparently
in a different ratio. In accord with this, males of the two species appear to respond optimally
to blends similar to those produced by their respective females: 1:100 for M. japona males
and 20:1 for M. longipes males (Tables 1 and 2). Although single-component blends that
are attractive to the other species were tested in the respective habitats of the two species,
no M. japona males approached lures in the habitat of M. longipes, and no M. longipes
males were captured in traps in the test for M. japona males. This result suggests that
geographical overlap of the species is not common (at least in the areas tested), and
precludes any possible hybridization of the two species. However, if there were any
geographical overlap, our data suggest that there would be little, if any, cross attraction
between the species.
Although E2,Z13-18:OAc was not detected in the pheromone glands of the two
Macroscelesia species, we were interested in its activity with respect to males of the two
species. The acetate appeared to have little effect on M. longipes males. However, in the
case of male M. japona, addition of this compound to E2,Z13-18:Ald gave increased trap
captures relative to the latter compound alone. Field Tests II and IV indicated that E2,Z13-
18:OAc may have acted as a substitute for E2,Z13-18:OH, the minor pheromone
component of M. japona. Substitution of a minor component by other analogs has been
observed in other lepidopteran species (Linn et al., 1984; Sugie et al., 2003; Inomata et al.,
2005). However, in field Test II, the blends of the aldehyde and acetate caught significantly
greater numbers of moths than the natural blends of the aldehyde and alcohol, with trap
catches to the latter blends being completely suppressed. This preferential response to an
unnatural blend over a natural one is perplexing, and suggests that M. japona males could
be attracted to another species that produces a blend of E2,Z13-18:Ald and E2,Z13-18:
OAc. To date, no species using this mixture as a sex pheromone has been identified, and the
mixture in our field trials attracted no other species. However, considering the diversity of
Sesiidae, and the apparent limited variety of chemicals used by this family as pheromone
components, it is possible that some species secrete such a mixture. Further studies on a
greater number of species of Sessiidae are necessary to identify such species.
Acknowledgments We are grateful to Mr. R. Wakasugi, D. Wakatsuki, S. Onodera, and students of the
Laboratory of Zoology at Meijo University, and Mr. T. Takamura in Nagoya City for collecting galls of
Macroscelesia spp., Dr. F. Mochizuki of Shin-Etsu Chemical Co., Ltd. for supplying dienyl acetate. We also
thank Drs. A. Mochizuki and K. Ito at NIAES, T. Nakazawa at BASE, and Mr. K. Fukuzumi in Nagoya City
for advice and information. This work was supported in part by a research fund from Heart Co. Ltd. (Tokyo,
Japan).
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