Parthenogenesis, calling behavior, and insect-released volatiles of leafminer moth Phyllonorycter emberizaepenella.

Article abstract of DOI:10.1023/A:1016233817754

We proved that the leafminer moth Phyllonorycter emberizaepenella (Lepidoptera: Gracillariidae) reproduces by parthenogenesis of the thelytoky type. Despite a complete absence of males, parthenogenetically reproducing females diurnally demonstrated the calling posture normally used for releasing signaling compounds. Two compounds, which we collected from a calling female, were identified as potential sex pheromone components: (8E, 10E)-8,10-tetradecadienyl acetate and (8E,10E)-8,10-tetradecadienol, the latter occurring only in trace amounts. In field experiments, no males were attracted to traps baited with either the potential sex pheromone or with virgin females. Both the pattern of behavior and the chemical characteristics of the pheromone of Ph. emberizaepenella species were similar to those known for Lepidoptera with the usual amphimictic mode of reproduction. Theoretical speculations that in thelytoky, where there is no need to find a sexual partner, the individuals would obtain certain advantages due to reduction in their sexual behavior, were, thus, not confirmed for Ph. emberizaepenella.

Full text of DOI:10.1023/A:1016233817754

Journal of Chemical Ecology, Vol. 28, No. 6, June 2002 (�C 2002)
PARTHENOGENESIS, CALLING BEHAVIOR, AND
INSECT-RELEASED VOLATILES OF LEAFMINER MOTH
Phyllonorycter emberizaepenella
¯
1�,2
¯
VINCAS BUDA, ILME LIBLIKAS,
1
2
RAIMONDAS MOZURAITIS,
2
2
CARL RIKARD UNELIUS, and ANNA-KARIN BORG-KARLSON
1Laboratory of Chemical Ecology
Institute of Ecology
Akademijos 2, LT-2600 Vilnius, Lithuania
2Department of Chemistry, Organic Chemistry
Royal Institute of Technology
SE-100 44 Stockholm, Sweden
(Received May 1, 2001; accepted February 16, 2002)
Abstract—We proved that the leafminer moth Phyllonorycter emberizaepenella
Lepidoptera: Gracillariidae) reproduces by parthenogenesis of the thelytoky
(
type. Despite a complete absence of males, parthenogenetically reproducing
females diurnally demonstrated the calling posture normally used for releas-
ing signaling compounds. Two compounds, which we collected from a calling
female, wereidentifiedaspotentialsexpheromonecomponents:(8E,10E)-8,10-
tetradecadienyl acetate and (8E,10E)-8,10-tetradecadienol, the latter occurring
only in trace amounts. In field experiments, no males were attracted to traps
baited with either the potential sex pheromone or with virgin females. Both
the pattern of behavior and the chemical characteristics of the pheromone of
Ph. emberizaepenella species were similar to those known for Lepidoptera with
the usual amphimictic mode of reproduction. Theoretical speculations that in
thelytoky, where there is no need to find a sexual partner, the individuals would
obtain certain advantages due to reduction in their sexual behavior, were, thus,
not confirmed for Ph. emberizaepenella.
Key Words—Thelytoky, diurnal rhythm, communication behaviour, (8E,10E)-
8
,10-tetradecadienyl acetate, (8E,10E)-8,10-tetradecadienol, Lepidoptera,
Gracillariidae.
�
To whom correspondence should be addressed. E-mail: Raimozu@julius.ktl.mii.lt
1
191
0
098-0331/02/0600-1191/0 �C 2002 Plenum Publishing Corporation

¯
1
192
MOZURAITIS ET AL.
INTRODUCTION
The parthenogenetic mode of reproduction in animals has a number of drawbacks
and advantages when compared with amphimixis. One potential advantage is the
absence of mate finding (Gilyarov, 1982; Went, 1984). However, from the view-
point of the theory of optimality (Rozen, 1969), for altering the genetic program
(including that of sex pheromone production and calling behavior), certain energy
expenses are needed, and these are justified only when there will be a benefit greater
than the cost of carrying out these modifications. With this in mind, the detailed
investigation of both chemical and behavioral elements related to “searching” for
a sexual partner in parthenogenetically reproducing animals is of interest.
Insects are convenient objects for such studies, especially species that use
chemical communication while locating their mates. The aim of this study was to
verify the parthenogenetic reproduction of the widespread leafminer moth Phyl-
lonorycter emberizaepenella that was reported earlier (B u¯ da et al., 1991) but never
confirmed, as well as to elucidate the sexual behavior of this moth and to determind
whether it releases potential sex pheromones during the calling.
METHODS AND MATERIALS
Insects
Common honeysuckle shrubs, Lonicera xylosteum L. leaves with Ph. ember-
izaepenella (Z.) (Lepidoptera, Gracillariidae) mines were collected at the end of
October 1989 and 1996–1998 and held in an outdoor container on moistened peat
ˇ
moss until the following spring. Specimens were collected from Vilnius (Zemieji
Paneriai) and from four other localities in Lithuania. The maximum distance be-
tween collection sites was 300 km. In addition, nondiapausing individuals were
collected at the beginning of June 1999. The leaves with pupae were placed in
28-ml glass vials, three mines per vial, and held under an artificial 16L:8D pho-
toperiod. A daylight lamp of 1000 W (type DRF, for greenhouses) was used. The
temperature was maintained nearly constant during the scotophase, with irregular
�
fluctuations within the range 14 � 1 C. Within 2 hr of the start of the photophase,
�
it was gradually raised to 20 � 1 C, kept constant until 2 hr before the end of
�
the photophase, and then gradually decreased, reaching 14 C when the next sco-
tophase began. Following emergence, the adults were collected daily just after the
onset of the photophase, then sexed and placed into individual holding vials (28-ml
volume), and provided with a solution of 5% of sucrose in water.
Parthenogenetic Mode of Reproduction
Three 2-day-old virgin females of Ph. emberizaepenella were placed in indi-
vidual containers together with green branches of the honeysuckle and were left to

LEAFMINER MOTH CHARACTERISTICS
1193
oviposit for one day. The branches were steeped in Knop’s solution (Grodzinskii
and Grodzinskii, 1973). This food source was sufficient for the complete develop-
ment of the progeny.
Diurnal Rhythm of Female Calling Behavior
At emergence, 32 virgin females were placed in individual glass vials and their
behavior was observed over a four-day period. During experiments, females were
provided with a solution of 5% of sucrose in water. Observations were made every
30 min during the last 2 hr of the scotophase until the 8th hour in the photophase,
and hourly during the remaining period of a photocycle. Observations during the
scotophase were made using a 4.5-V red lamp light. Females were classified as
calling when the abdomen was raised clearly above the wings (Figure 1B).
Collection of Compounds
Compounds released from calling females were collected using the solid
phase microextraction (SPME) technique (Borg-Karlson and Mozuraitis, 1996).
�
The SPME fiber (100-mm polydimethylsiloxane) was purified at 225 C for about
10 min in a GC injector before being placed a few millimeters away from the
abdominal tip of a calling female for 2–4 hr at the beginning of the calling period.
Then the SPME fiber was inserted in a glass tube (1.75-ml volume), containing
undecyl acetate (10 ng) and pentadecyl acetate (100 ng) on the inner wall as internal
�
standards, and the sorption continued for another 15 min at 22 C. The volatiles
from 12 females were collected individually during their calling and noncalling
periods and analyzed one by one in this way.
Chemical Analyses
The signaling compounds were identified by using the Finnigan SSQ 7000
GC-MS system, including a Varian 3400 GC. One DB-5 and one DB-Wax silica
capillary column (30 m � 0.25 mm ID, 0.25-�m film thickness) were used with
�
�
�
�
�
a temperature program of 70 C (1 min), increased by 10 C/min to 140 C, then
�
�
�
�
by 1 C/min up to 150 C, by 2 C/min up to 170 C, by 5 C/min up to 200 C, and
�
thereafter maintained constant at 200 C for 17 min. The split/splitless injector
temperature was 225 C, and the splitless period was 30 sec. Helium was used as
�
the carrier gas with an inlet pressure of 5 psi. Electron ionization mass spectra
�
were determined at 70 eV with the ion source at 150 C. When the sample had
been injected, the desorption of the sex pheromone components and of the inter-
nal standards from the SPME fiber was checked by subsequent injection into a
split/splitless injector of a second GC for 30 sec of splitless time at an injector
�
temperature of 225 C. Total desorption of the compounds occurred during the first
injection.

¯
1
194
MOZURAITIS ET AL.
FIG. 1. Postures of virgin Phyllonorycter emberizaepenella females: (A) resting,
B) calling.
(
The volatiles collected from females during their calling and noncalling peri-
ods were compared chromatographically, and only those coming exclusively from
calling periods were analyzed. The compounds selected for analysis were iden-
tified by comparison of their mass spectral data and GC-retention times with the
corresponding data of synthetic standards.
Chemicals
All isomers of 10,12-tetradecadienols and their acetates used as GC-MS stan-
dardswereavailablefrompreviousresearch, whereasthe8,10-tetradecadienolsand

LEAFMINER MOTH CHARACTERISTICS
1195
their acetates were synthesized at the Department of Chemistry, Organic Chem-
istry, The Royal Institute of Technology, Stockholm. The synthetic intermediates
as well as the products were characterized with a Bruker AM 400 spectrometer
(
NMR spectra were recorded in CDCl3), and the GC-MS system is described in
the section above (Chemical Analyses).
-Bromooctanol (2; Figure 2) was prepared according to Rossi et al. (1981),
8
Gardette et al. (1983), and Tellier (1990). 8-Hydroxyoctyl-triphenylphosphonium
bromide (3; Figure 2) was synthesized as in Vinczer et al. (1987). (8E,10E)-8,10-
tetradecadienol (4; Figure 2) and (8Z, 10E)-8,10-tetradecadienol (5; Figure 2)
were made according to Lo and Shiao, (1986). The products were obtained in a
mixture of 4 (40%) and 5 (60%). ZZ and EZ isomers were not detected by GC-MS.
Isomers 4 and 5 were separated by chromatography on silica gel impregnated with
15% AgNO3. Finally, 99.0% isomerically pure 4 was derived from a urea inclusion
complex, and 5 was obtained in 99.2% purity by crystallization from pentane at
�
�
30 C.
1
(
8E,10E)-8,10-Tetradecadienol 4. H NMR: �: 5.97 (m, 2H, CH CH-
CH CH ), 5.54 (m, 2H, CH CH-CH CH ), 3.61 (t, J = 6.6 Hz, 2H, CH2-
OH), 2.02 (m, 4H, CH2-CH ), 1.24–1.54 (m, 12H), 0.89 (t, J = 5.7 Hz,
+
3
H, CH2-CH3). EI-MS [m/z (relative intensity)]: 210 (M , 34), 192 (6), 135
(
(
(
11), 121 (18), 111 (7), 110 (14), 109 (17), 108 (7), 107 (12), 98 (10), 97 (10), 96
45), 95 (32), 94 (13), 93 (24), 91 (8), 83 (9), 82 (41), 81 (77), 80 (22), 79 (42), 77
8), 69 (9), 68 (26), 67 (100), 55 (32), 54 (34), 43 (12), 41 (30).
1
(
8Z,10E)-8,10-Tetradecadienol 5. H NMR: �: 6.28 (dd, J = 11.0, 14.8 Hz,
8
9
10
11
8
1
H, C H C H-C H C H ), 5.93 (dd, J = 10.9, 11.0 Hz, 1H, C H
9 10 11 8 9 10 11
C H-C H C H ), 5.63 (dt, J = 7.3, 15.0 Hz, 1H, C H C H-C H C H ),
8
9
10
11
5
2
7
1
.31 (dt, J = 7.4, 10.8 Hz, 1H, C H C H-C H C H ), 3.61 (t, J = 6.5 Hz,
H, CH2-OH), 2.06-2.16 (m, 4H, CH2-CH ), 1.28-1.65 (m, 12H), 0.89 (t, J =
+
.2 Hz, 3H, CH2-CH3). EI-MS [m/z (relative intensity)]: 210 (M , 34), 192 (6),
35 (8), 122 (7), 121 (18), 111 (7), 110 (15), 109 (17), 108 (7), 107 (13), 98
(10), 97 (10), 96 (43), 95 (33), 94 (13), 93 (25), 91 (7), 83 (9), 82 (38), 81 (75),
8
4
0 (21), 79 (38), 77 (8), 69 (10), 68 (25), 67 (100), 55 (31), 54 (33), 43 (12),
1 (28).
(8E,10E)-8,10-Tetradecadienyl Acetate (6; Figure 2). Pyridine (2 ml) and
acetic anhydride (0.5 ml) were added to compound 4 (0.200 g, 0.95 mmol) at
room temperature, and the mixture was left in a refrigerator overnight. The next
day it was poured into ice-water and extracted with hexane. The extract was dried
and chromatographed to yield 6 (0.205 g, 91%). No loss in isomeric purity was ob-
1
served. H NMR: �: 5.97 (m, 2H, CH CH-CH CH ), 5.54 (m, 2H, CH CH-
CH CH ), 4.01 (t, J = 6.5 Hz, 2H, CH2-COO ), 2.08 (m, 4H, CH2-CH ),
2
.04 (s, 3H, CH3-COO ), 1.22-1.50 (m, 12H), 0.88 (t, J = 5.6 Hz, 3H, CH2-
+
CH3). EI-MS [m/z (relative intensity)]: 252 (M , 44), 192 (33), 163 (7), 149 (14),
136 (9), 135 (27), 124 (7), 123 (7), 122 (14), 121 (40), 110 (28), 109 (21), 108 (16),

¯
1
196
MozUraitis et al.
Fig. 2. Schemes of the syntheses of 8,10-tetradecadienols and their acetates.

LEAFMINER MOTH CHARACTERISTICS
1197
1
8
4
07 (22), 97 (8), 96 (44), 95 (33), 94 (23), 93 (38), 91 (8), 83 (7), 82 (38), 81 (72),
0 (35), 79 (7), 78 (8), 69 (10), 68 (22), 67 (100), 66 (8), 55 (28), 54 (24), 43 (51),
1 (24).
(8Z,10E)-8,10-Tetradecadienyl Acetate (7; Figure 2). Acetylation of 5, as
described above for 4, gave 7 in 88% yield. Minor isomerization products were
separated by liquid chromatography on AgNO3/silica gel to yield 99% isomeri-
1
8
9
cally pure acetate 7. H NMR: �: 6.28 (dd, J = 11.0, 14.8 Hz, 1H, C H C H-
10
11
8
9
10
11
C H C H ), 5.93 (dd, J = 10.9, 11.0 Hz, 1H, C H C H-C H C H ),
8
9
10
11
5
1
2
.65 (dt, J = 7.3, 15.0 Hz, 1H, C H C H-C H C H ), 5.30 (dt, J = 7.4,
8 9 10 11
0.8 Hz, 1H, C H C H-C H C ), 4.02 (t, J = 6.6 Hz, 2H, CH2-COO ),
.10 (m, 4H, CH2-CH ), 2.03 (s, 3H, CH3-COO ), 1.26–1.40 (m, 12H), 0.90
+
(
t, J = 6.8 Hz, 3H, CH2-CH3). EI-MS [m/z (relative intensity)]: 252 (M , 37),
1
92 (33), 163 (7), 149 (18), 136 (9), 135 (25), 124 (7), 122 (15), 121 (42), 110
(29), 109 (19), 108 (15), 107 (22), 97 (8), 96 (43), 95 (35), 94 (25), 93 (38), 91 (7),
8
6
3 (8), 82 (42), 81 (77), 80 (35), 79 (63), 78 (7), 77 (8), 69 (9), 68 (23), 67 (100),
6 (7), 55 (31), 54 (26), 43 (53), 41 (28).
8
-Bromo-1-(2-tetrahydropyranyloxy)-octane (8; Figure 2) was synthe-
sized as described by Miyashita et al. (1977). 8-(2-Tetrahydropyranyloxy)-octanal
9; Figure 2) was made based on Ando et al. (1988). 4-(E)-10-(2-Tetrahydro-
pyranyloxy)-2-decenal(10, Figure 2) was made according to Ando et al. (1985).
8E,10Z)-1-(2-Tetrahydropyranyloxy)-8,10-tetradecadiene (11, Figure 2) was
synthesized based on the methods published by Vinczer et al. (1988).
(
(
(
8E,10Z)-8,10-Tetradecadienol (12; Figure 2). Deprotection of 11 (2.2 g,
�
7
.5 mmol) was performed in EtOH (25 ml) at 55 C for 6 hr in the presence of pyri-
dinium tosylate (188 mg, 0.75 mmol) as a catalyst. Evaporation of the ethanol and
chromatography yielded a crude product from which 0.86 g (55%) 97% isomeri-
cally pure 12 was obtained after crystallization from pentane. Chromatography
on AgNO3-impregnated silica gel enriched the isomeric purity up to 99.5%.
1
8
9
10
11
H NMR: �: 6.32 (dd, J = 10.9, 15.0 Hz, 1H, C H C H-C H C H ), 5.95
8
9
10
11
(
dd, J = 10.6, 10.9 Hz, 1H, C H C H-C H C H ), 5.64 (dt, J = 7.0, 15.1
8 9 10 11 8 9
Hz, 1H, C H C H-C H C H ), 5.30 (dt, J = 7.5, 10.9 Hz, 1H, C H C H-
10
11
C H C H ), 3.63 (t, J = 6.9 Hz, 2H, CH2-OH), 1.90–2.22 (m, 4H, CH2-
CH ), 1.21–1.72 (m, 12H), 0.93 (t, J = 7.6 Hz, 3H, CH2-CH3). EI-MS [m/z
+
(relative intensity)]: 210 (M , 49), 192 (13), 176 (13), 167 (8), 135 (9), 125 (8),
1
9
23 (17), 122 (7), 121 (25), 110 (17), 109 (23), 107 (15), 98 (9), 97 (18), 96 (47),
5 (35), 94 (24), 93 (19), 91 (17), 87 (10), 85 (12), 83 (8), 82 (44), 81 (82), 80
(
24), 79 (64), 71 (23), 70 (7), 69 (15), 68 (24), 67 (100), 66 (9), 55 (25), 54 (32),
1 (17).
8E,10Z)-8,10-Tetradecadienyl acetate (13; Figure 2). The alcohol 12 was
acetylated in the manner described above for compounds 6 and 7. Acetylation gave
3 in 87% yield. Minor isomerization products were separated by chromatogra-
phy on AgNO3-impregnated silica gel to yield 99.5% isomerically pure acetate
4
(
1

¯
1
198
MOZURAITIS ET AL.
1
8
9 10 11
1
5
7
3. H NMR: �: 6.32 (dd, J = 10.9, 15.0 Hz, 1H, C H C H-C H C H ),
8 9 10 11
.95 (dd, J = 10.6, 10.9 Hz, 1H, C H C H-C H C H ), 5.64 (dt, J =
8
9
10
11
.0, 15.1 Hz, 1H, C H C H-C H C H ), 5.30 (dt, J = 7.5, 10.9 Hz, 1H,
8
9
10
11
C H C H-C H C H ), 4.05 (t, J = 6.8 Hz, 2H, CH2-COO ), 1.90–2.16
(
m, 4H, CH2-CH ), 2.04 (s, 3H, CH3-COO ), 1.30–1.70 (m, 12H), 0.91 (t,
+
J = 7.3 Hz, 3H, CH2-CH3). EI-MS [m/z (relative intensity)]: 252 (M , 39),
1
(
8
(
92 (32), 149 (12), 136 (8), 135 (26), 123 (7), 122 (14), 121 (40), 110 (28), 109
19), 108 (15), 107 (21), 97 (7), 96 (42), 95 (32), 94 (20), 93 (36), 91 (8), 83 (7),
2 (36), 81 (72), 80 (34), 79 (58), 69 (8), 68 (21), 67 (100), 55 (30), 54 (24), 43
52), 41 (27).
-Bromoheptanol (15; Figure 2) was obtained by a procedure similar to
7
the one described above for 8-bromooctanol, 2. For 7-Bromo-1-(2-tetrahydro-
pyranyloxy)-heptane (16; Figure 2), the alcohol 15 was protected as its THP–ether
1
by the usual method, which gave 16 in 95% yield. H NMR: �: 4.57 (t, J = 3 Hz,
1
H), 3.31–3.86 (m, 4H, CH2-O ), 3.63 (t, J = 6.6 Hz, 2H, CH2Br), 1.30–
1.81 (m, 16H). 1-(2-Tetrahydropyranyloxy)-8-nonyne (17; Figure 2) was prepared
according to Rossi et al. (1981); Gardette et al. (1983).
-Bromo-1-pentyne (19; Figure 2). Potassium hydroxide (67.2 g, 1.2 mol)
1
was dissolved in water (370 ml). Bromine (32 g, 0.2 mol) was added dropwise at
�
0
C during 30 min, and 1-pentyne (18) (13.0 g, 0.19 mol) was then added to the
�
solution, while the temperature was maintained at 0 C. The reaction mixture was
stirred for 14 hr at room temperature. The product was extracted with a pentane–
diethyl ether mixture (1:1), and the extract was dried and concentrated. Distillation
1
gave 23.9 g of 19, an 85% yield. H NMR: �: 2.18 (t, J = 7.0 Hz, 2H, CH2-C ),
1
.54 (td, J = 7.3, 14.3 Hz, 2H, CH2 ), 0.98 (t, J = 7.4 Hz, 3H, CH3).
1-(2-Tetrahydropyranyloxy)-8,10-tetradecadiyne (20; Figure 2) was obtained
by a procedure described by Svirskaya et al. (1980); Chisholm et al. (1985);
De Jarlais and Emken (1987). (8Z,10Z)-1-(2-Tetrahydropyranyloxy)-8,10-
tetradecadiene (21; Figure 2) was synthesized according to Svirskaya et al. (1980);
Chisholm et al. (1985); De Jarlais and Emken, (1987).
(8Z,10Z)-8,10-Tetradecadienol (22; Figure 2). Compound 21 (1.78 g,
6
.1 mmol) was added to pyridinium tosylate in ethanol (25 ml). The mixture
�
was warmed at 55 C for 4 hr. Evaporation of the ethanol, followed by chromatog-
raphy, yielded 1.2 g (80%) of 22 with an isomeric purity of 97%. After crystal-
�
1
lization from pentane at � 20 C, no isomers were detected by GC. H NMR: �:
6
.28 (m, 2H, CH CH-CH CH ), 5.47 (m, 2H, CH CH-CH CH ), 3.65
(
t, J = 6.7 Hz, 2H, CH2-O ), 2.18 (m, 4H, CH2-CH ), 1.56 (m, 2H), 1.26–
1
2
1
.46 (m, 10H), 0.94 (t, J = 7.3 Hz, 3H, CH3). EI-MS [m/z (relative intensity)]:
+
10 (M , 26), 192 (8), 135 (12), 122 (7), 121 (19), 111 (8), 110 (18), 109 (15),
08 (8), 107 (12), 98 (12), 97 (11), 96 (45), 95 (34), 94 (14), 93 (25), 91 (7), 83
(9), 82 (42), 81 (84), 80 (22), 79 (42), 77 (8), 69 (10), 68 (27), 67 (100), 55 (33),
54 (35), 43 (11), 41 (31).

LEAFMINER MOTH CHARACTERISTICS
1199
(8Z,10Z)-8,10-Tetradecadienyl acetate (23; Figure 2). The acetylation proce-
dure described above gave the acetate 23 in 92% yield. No isomerization occurred
1
during the acetylation. H NMR: �: 6.28 (m, 2H, CH CH-CH CH ), 5.56
(
m, 2H, CH CH-CH CH ), 4.08 (t, J = 6.7 Hz, 2H, CH2-O ), 2.17 (m, 4H,
CH2-CH ), 2.08 (s, 3H, CH3COO ), 1.34–1.67 (m, 12H), 0.94 (t, J = 7.4 Hz,
+
3
1
H, CH3). EI-MS [m/z (relative intensity)]: 252 (M , 24), 193 (8), 192 (43),
63 (7), 149 (13), 136 (10), 135 (26), 124 (6), 123 (7), 122 (14), 121 (39), 110
(27), 109 (18), 108 (16), 107 (22), 97 (8), 96 (40), 95 (35), 94 (24), 93 (40), 91
(9), 83 (8), 82 (40), 81 (81), 80 (35), 79 (66), 78 (7), 77 (8), 69 (11), 68 (23), 67
(100), 66 (8), 55 (32), 54 (37), 43 (54), 41 (28).
Trapping Tests
The trapping tests using virgin calling females were carried out in the vicinity
of Vilnius in eastern Lithuania from April 20 to May 20, 1990. Two-day-old virgin
calling females were placed in a 30-ml wire mesh cylinder containing a sponge
with a solution of 5% of sucrose in water that was replaced every two days.
The container was placed in an opaque white Atracon A delta trap containing
an exchangeable bottom (11 � 18 cm), coated with Pestifix glue. Ten traps, each
containing one female, were placed about 1 m above the ground on a branch of
common honeysuckle shrub with a distance of >10 m between traps and inspected
daily. Females were replaced by young females when they reached age 10 days of
or were found to be not calling.
The field tests of potential synthetic sex pheromone components were carried
out at the same locality in the vicinity of Vilnius from May 19 to June 15, 2000. The
compounds were dissolved in hexane (Merck p.a.) and, either alone or in binary
mixtures, soaked from the inside into the walls of red rubber tube dispensers
(
1
18 � 15 mm). Samples of 200 �g 8E,10E–14:OAc/dispenser; 0.2 �g 8E,10E–
4:OH/dispenser; 200 �g 8E,10E–14:OAc + 0.2 �g 8E,10E–14:OH/dispenser
and 50 �l hexane/dispenser (as a control) were tested. Five replications of each
lure were used. In order to diminish the impact of biotopic variation on the trapping
results, all traps of one replication set were moved to the location of traps of another
replication set every five days.
RESULTS
Biological Characteristics of Ph. emberizaepenella Sample. After the winter
diapause, 172 moths were obtained from pupae collected in the autumn of 1989
(
42% of the pupae were infected by parasites and 12% died due to other causes). In
all, 748 moths emerged from the mined leaves collected in the autumns of 1996–
998, and 82 moths emerged from the pupae collected in the summer of 1999.
All of the adult moths were females. In addition to the individuals obtained from
1

¯
1
200
MOZURAITIS ET AL.
pupae, we collected 134 moths outdoors from April 20 through May 20, 1990 with
an entomological net, and even in this group all specimens were females. Under
laboratory conditions the average length of life of the adult female was 15 � 5 days
(
N = 37).
Parthenogenetic Mode of Reproduction. Normally developed caterpillars
emerged from unfertilized eggs laid by virgin females of Ph. emberizaepenella.
Host plant twigs with leaves containing offspring of three Ph. emberizaepenella
females remained suitable as food sources for the larvae long enough to allow
them to form typical pupae. Seventeen adults of the F1 generation were obtained
from the pupae, and all of them were females.
Calling Behavior of Females. By observing the behavior of virgin females in
nature, we established that they preferred to stay on the underside of the host plant
leaves, where they remained stationary, while exhibiting the calling posture. The
most typical elements of this posture were the following: wings slightly spread and
lowered, abdominal tip dorsally curved and distal abdominal segments protruded,
and antennae placed along the wings (Figure 1B).
Under laboratory conditions, females started to call on the second day after
emergence. The peak of activity was registered 0.5–3 hr after the beginning of the
light phase. Nearly 100% of the females (N = 37) were active during the calling
peak. The total duration of the active period within the group of 2- to 4-day-old
females was about 6 hr/day (Figure 3).
Chemical Identification of Compounds Collected from Calling Females.
Two compounds were found to be specific for the calling periods of virgin
FIG. 3. Diurnal rhythm of calling activity of virgin Phyllonorycter emberizaepenella
females under laboratory conditions. Shaded areas: scotophase, white area: photophase.
The females were not found calling during the first day after emergence. N = 32 females
in each day of the observation.

LEAFMINER MOTH CHARACTERISTICS
1201
Ph. emberizaepenella females, when GC-MS chromatograms from calling and
noncalling periods were compared (Figure 4AB). One of these compounds showed
m/z 61 but not m/z 60, typical diagnostic ions of acetates and fatty acids, respec-
tively. The diagnostic ion, m/z 192 (M-60), indicated di-unsaturation and a chain
+
.
length of C14. In addition, the well-expressed molecular ion m/z 252 (M ) con-
firmed that the compound was a tetradecadienyl acetate and indicated that the
double bonds were conjugated (Figure 5A).
The second compound showed a fragmentation pattern similar to that of the
tetradecadienyl acetate. The presence of the diagnostic ions m/z 31 and m/z 210
+ +.
.
(
M ) instead of m/z 61 and m/z 252 (M ), respectively, indicated that the second
compound was a tetradecadienol (Figure 5B).
In all unsaturated sex pheromone components identified from species of the
genus Phyllonorycter, double bonds occurred only at even positions (Moz u¯ raitis
et al., 1999; Arn et al., 2001). Therefore, we compared the retention times of the
two compounds collected from the calling females with those of synthetic 8,10-
and 10,12-tetradecadienols and their acetates. This comparison showed that the
volatiles released by the Ph. emberizaepenella females were 8E,10E–14:OAc and
8
E,10E–14:OH (Table 1). A comparison of the mass spectra of the compounds
collected from calling females with those of synthetic standards confirmed the
results of the GC analyses.
Volatiles from all 12 calling females contained a trace amount of 8E,10E–
1
4:OH relative to the acetate, detected only by using the most abundant diagnostic
+
.
ions m/z 67, m/z 81 and m/z 210 (M ) (Figure 5B).
Trapping Experiments. During the trapping period, which was about twice
as long as the average lifetime of adult females, neither traps containing virgin
calling females nor lures made up with potential sex pheromones captured any
males, in spite of the large number of moths of this species in the habitat. These
facts indicated that the probability of finding a male among moths of the first
generation of this population was extremely small, close to zero. Consequently,
8
E,10E–14:OAc and the corresponding alcohol have to be considered as potential
sex pheromone components of this leaf-miner species.
DISCUSSION
The development of adults from unfertilized eggs, as well as the absence
of males in both generations, confirmed the previous report (B u¯ da et al., 1991)
on parthenogenesis of Ph. emberizaepenella species. When only females develop
from unfertilized eggs, the term thelytoky is applicable for characterizing the
parthenogenesis. Regular thelytoky is rare among the Lepidoptera, but it has
been found in some species belonging to the families Psychide, Tineidae, and
Lymantridae (reviews: Cockayne, 1938; Suomalainen, 1950; Went, 1984). As far

¯
1
202
MOZURAITIS ET AL.
FIG. 4. Total and selected ion chromatogram records of volatiles collected from one
Phyllonorycter emberizaepenella female. (A) Total ion chromatogram in the range m/z
3
0–400; Noncalling and calling indicate that volatiles were obtained during noncalling
and calling periods of a female, respectively; DB-5 fused silica capillary column (30 m �
.25-mm ID, 0.25-�m film thickness). (B) Selected ion chromatograms where m/z 67 and
0
m/z 81 ion fragments indicate unsaturation; m/z 210 is the molecular ion characteristic
of tetradecadienols; Total represents chromatogram in the ion range m/z 30–400; HP-Wax
fused silica capillary column (30 m � 0.25 mm ID, 0.25-�m film thickness); m/z indicates
the ratio of the mass of the ion to the number of charges on it. The numbers at the upper-
right corner of the chromatograms, for example, E+05 5.21, represent the number of counts
5
related to the abundance of the ions formed and E+05 5.21 has to be read as 5.21� 10 .

LEAFMINER MOTH CHARACTERISTICS
1203
FIG. 5. Mass spectra of the compounds obtained only during calling periods of Phyllono-
rycter emberizaepenella females: (A) 8E,10E-14:OAc; (B) 8E,10E-14:OH; m/z indicates
+
.
the ratio of the mass of the ion to the number of charges on it; M represents the molecular
ion; the number at the upper-right corner of the mass spectrum, e.g., E+0.5 1.29, represents
the number of counts related to the abundance of the ions formed and E+0.5 1.29 has to be
5
read as 1.29 � 10 .
as we know, Ph. emberizaepenella is the first case of thelytokous parthenogenesis
found in the Gracillariidae family.
Ph. emberizaepenella females demonstrated a calling posture similar to that
of other species of the same genus that we have investigated: Ph. ulmifoliella
(Hb.) (Moz u¯ raitis et al., 1997), Ph. acerifoliella (Z.) and Ph. heegerella (Z.)
(Moz u¯ raitis et al., 2000), and Ph. sorbi (Frey.) and Ph. strigulatella (Lien et Z.)
(Moz u¯ raitis, 2000). This posture is also similar to that of the pyralids, Ephestia
(=Anagasta) kuehniella (Z.) (Traynier, 1968) and Plodia interpunctella (Hb.)
(Brady and Smithwich, 1968).

¯
1
204
MOZURAITIS ET AL.
TABLE 1. RETENTION TIMES OF COMPOUNDS OBTAINED FROM CALLING Phyllonorycter
enberizaepenella FEMALES, AND SYNTHETIC 8,10- AND 10,12-TETRADECADIENOLS AND
THEIR ACETATES, WHEN PASSING THROUGH TWO CAPILLARY COLUMNS
Retention time (min:sec)
Compound
Polar column (HP-wax) Unpolar column (DB-5)
From female
(
(
8E,10E)-8,10-tetradecadienyl acetate
8E,10E)-8,10-tetradecadienol
39:10
42:14
34:05
26:03
Synthetic
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
8Z,10Z)-8,10-tetradecadienyl acetate
38:44
37:24
38:08
39:10
41:48
40:51
41:19
42:14
40:18
39:23
40:05
39:47
42:53
42:11
42:43
42:30
33:46
32:38
33:10
34:05
25:35
23:49
24:30
26:02
34:39
33:46
34:31
34:18
26:43
25:35
26:31
26:15
8Z,10E)-8,10-tetradecadienyl acetate
8E,10Z)-8,10-tetradecadienyl acetate
8E,10E)-8,10-tetradecadienyl acetate
8Z,10Z)-8,10-tetradecadienol
8Z,10E)-8,10-tetradecadienol
8E,10Z)-8,10-tetradecadienol
8E,10E)-8,10-tetradecadienol
10Z,12Z)-10,12-tetradecadienyl acetate
10Z,12E)-10,12-tetradecadienyl acetate
10E,12Z)-10,12-tetradecadienyl acetate
10E,12E)-10,12-tetradecadienyl acetate
10Z,12Z)-10,12-tetradecadienol
10Z,12E)-10,12-tetradecadienol
10E,12Z)-10,12-tetradecadienol
10E,12E)-10,12-tetradecadienol
A distinct diurnal rhythm of the calling behavior was established on the basis
of the calling posture. Ph. emberizaepenella females were active after the start of
the light period of a day, which is a common calling time for other species of the
samegenus[Ph. ulmifoliella(Moz u¯ raitisetal., 1997);Ph. ringoniella(Matsumura)
(Boo and Jung, 1998); Ph. acerifoliella and Ph. heegerella, (Moz u¯ raitis et al.,
2000), Ph. sorbi and Ph. strigulatella (Moz u¯ raitis, 2000)] and for some species of
other families (Dreisig, 1986; McNeil, 1991, T o´ th et al., 1995).
Both compounds identified from calling Ph. emberizaepenella females were
new for the family Gracillariidae, but their chemical structures corresponded to
the general pattern of the structures determined for sex pheromones of other
Phyllonorycter species. These compounds were aliphatic C12–C14 straight-chain,
saturated, mono- or di-unsaturated acetates or alcohols, with Z or E double bonds
occurring in positions 4, 8, 10, and 12 (Moz u¯ raitis et al., 1999; Arn et al., 2001).
Assuming that both compounds were collected from virgin females only
during calling periods, and their chemical structures corresponded to the general
pattern of the ones determined for sex pheromones from other Phyllonorycter
species, it is possible that 8E,10E–14:OAc alone or in a binary mixture with a

LEAFMINER MOTH CHARACTERISTICS
1205
corresponding alcohol is a sex pheromone conponent of Ph. emberizaepenella
species.
All these characteristics of chemical communication of Ph. emberizaepenella
females are very similar or even identical to those of Lepidoptera with the usual
amphimictic mode of reproduction. In spite of the parthenogenetic reproduction
during which complete reductionof sexual behaviormight be expected, the females
of this leafmining species exhibited the calling posture and released potential sex
pheromone with the diurnal rhythm of such behavior that is inherent in the process.
Why do parthenogenetically reproducing females still call and synthesize and
release potential sex pheromone in the absence of males?
A reduction of sexual behavior involves changes in the physiology of an
organism. From the viewpoint of the theory of optimality (Rozen, 1969), these
changes are justified only when they are more beneficial than the cost of carrying
out all necessary modifications. The benefit of the absence of male finding would
include both resources and time saved from biosynthesis of a sex pheromone,
sex pheromone release, and exposure to potential natural enemies such as egg
parasitoids that use moth sex pheromones to locate resources. While the costs
include those for turning off sex pheromone biosynthesis and calling behavior.
The greatest benefit would be achieved by turning off both calling behavior
and biosynthesis of sex pheromones. Unfortunately, there are no data available on
these processes in the family Gracillariidae. Thus, our discussion must be based
on knowledge of other lepidopteran taxa.
In most investigated lepidopteran species, sex pheromone biosynthesis re-
quires pheromone biosynthesis activating neuropeptide (PBAN) or PBAN-like
compounds (Ramaswamy, 1997; Tillman et al., 1999). To turn off production
of a single peptide would not be complicated or costly if its role is not multi-
functional. However, in some species, pheromone glands are able to produce sex
pheromone through the diel period without the presence of PBAN, and this com-
petence is achieved as a result of developmental–metamorphic events (Tang et al.,
1989, 1991). The prediction of a mechanism and the cost of switching off sex
pheromone biosynthesis in these species is unclear.
Calling in Lepidoptera is usually controlled by neural outputs from the brain
or other neuronal centers (Sasaki et al., 1983; Itagaki and Corner, 1986; Tang et al.,
1987; Christensen et al., 1994). Thus, the stopping of calling behavior should not
be costly. However, more complex regulation of pheromone release occurs in two
migratory noctuid moth species. In the true armyworm moth, Pseudatelia unipunc-
tata (Haworth), juvenile hormone (JH) must be continuously present to maintain
calling behavior once initiated (Cusson and McNeil, 1989a), and the same hormone
is important for ovarian development (Cusson and McNeil, 1989b). The similar
function of JH has been demonstrated in black cutworm moth, Agrotis ipsilon
(Hufnagel) (Gadenne et al., 1993; Picombon et al., 1995). Turning off calling by
inhibiting production of JH would lead to termination of ovarian development, thus

¯
1
206
MOZURAITIS ET AL.
disturbing reproduction. In such an event, the cost for termination of calling and
sex pheromone production obviously would overcome the benefit, and it is most
likely that females would continue to release signaling compounds despite the
complete lack of males. Possibly, similar regulation of pheromone release might
occur in parthenogenetically reproducing Ph. emberizaepenella species.
The theoretical speculation that in thelytoky, individuals obtain advantages
due to a reduction in their sexual behavior when there is no need to find a sexual
partner clearly needs re-examination.
Acknowledgments—We thank Dr. G. Aulin-Erdtman for valuable discussions and language revi-
sion of the manuscript and two anonymous reviewers for valuable comments and kind suggestions. This
study was supported by the Agricultural and Forest Research Council (Sweden) and by the Swedish
Institute.
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