Journal of Chemical Ecology, Vol. 13, No. 7, 1987
SEX PHEROMONES OF RICE MOTH, Corcyra cephalonica STAINTON II. Identification and Role of Female Pheromone
D.R. HALL, 1 A. CORK, 1 R. LESTER, l B.F. N E S B I T T , ~ and P. Z A G A T T I 2 ITropical Development and Research Institute 56/62 Gray's Inn Road, London WC1X 8LU, England 21NRA-CNRS, Laboratoire des Mddiateurs Chimiques Dnmaine de Brou~ssy, Magny-les-Hameaux F-78470 St-R~my-lks-Chevreuse, France (Received June 13, 1986; accepted September 29, 1986) Abstract--Laboratory investigations of mating behavior in the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae; Galleriinae) showed that male moths are attracted at short range to live, virgin female moths and to female abdominal-tip extract. Volatiles collected from virgin female moths contained one component eliciting an electroantennographic (EAG) response from the male moth, and the chemical, spectroscopic, and chromatographic data on this component were consistent with that of synthetic 6,10,14-trimethyl-2-pentadecanol. This compound caused an EAG response from the male moth and attracted male moths in the bioassay. The pheromone is thought to play a role in courtship, and the synthetic material was shown to cause the male moths to search for a mate and attempt copulation. Key Words--Rice moth, Corcyra cephalonica, Lepidoptera, Pyralidae, Galleriinae, female pheromone, courtship pheromone, olfactometer, electroantenogram, 6,10,14-trimethyl-2-pentadecanol.
INTRODUCTION Studies o f m a t i n g b e h a v i o r in the rice m o t h , Corcyra cephalonica (Lepidoptera: Pyralidae; Galleriinae) h a v e s h o w n that f e m a l e m o t h s are attracted to m a l e moths, and m a l e m o t h s are attracted to f e m a l e m o t h s (Zagatti et al., 1987). The m a l e m o t h s w e r e f o u n d to p r o d u c e ( E , E ) - and ( Z , E ) - f a r n e s a l (3,7,11-trim e t h y l - 2 , 6 , 1 0 - d o d e c a t r i e n a l ) f r o m glands in the f o r e w i n g s , and the synthetic 1575 0098-0331/8710700-1575505.00/0 9 1987 Plenum Publishing Corporation
1576
HALL ET AL.
compounds were shown to attract walking female moths (Zagatti et al., 1987). This paper describes identification of the female-produced pheromone and studies to establish its role in the mating behavior of this species. METHODS AND MATERIALS
Insect Material. Laboratory cultures of C. cephalonica were maintained as described by Zagatti et al. (1987). The identification work in London was carfled out with moths from cultures of Malawi origin. Moths from cultures originating in India were used for the bioassay work in France. Pheromone Collection and Purification. For bioassay work, the abdominal tips (three posterior segments including ovipositor) of 38 virgin female moths were excised 2 hr into the scotophase. The tips were extracted in 1 ml hexane for 2 hr and the extract filtered through glass wool and reduced to 100 # under nitrogen. For the identification work, ovipositor or whole-body washes from virgin female moths were made under a variety of conditions: age 0-7days; at various times throughout a 12 hr-12 hr light-dark cycle or under constant low light; in heptan~, ether, or dichloromethane. To collect pheromone by entrainment, 20-30 virgin female moths were placed in a silanized, 3-liter, bolt-head flask with two paper tissues. Laboratory air was drawn at 1 liter/min into the bottom of the flask through a charcoal filter (12 x 2 cm; 10-18 mesh) and out through two glass filters (1.5 cm ID) packed with 2.5 g Porapak Q (50-80 mesh) purified by washing well with dichloromethane. Pheromone was collected continuously under natural lighting conditions at 20-25~ Twice per week, the Porapak filters were extracted with a total of 25 ml dichloromethane, and dead moths were replaced with freshly emerged ones. After seven weeks of entrainment, the Porapak filter extracts were combined and evaporated to 5 ml on a rotary evaporator at or below room temperature. Florisil (0.5 g, 100-200 mesh) was added and the remaining solvent removed on the rotary evaporator. The coated Florisil was deposited on the top of a column of 9.5 g Florisil (30 x 1 cm) made up in pentane. This was then eluted with 50-ml aliquots of diethyl ether-pentane mixtures containing 0, 2, 5, 10, 20, 30, 50, 75, and 100% ether. Fractions of 10 ml were collected. Gas Chromatography (GC). Columns packed with SE-30 or Carbowax 20 M and fused silica capillary columns coated with CPSil 5 CB or CP Wax 57CB were used as described in Zagatti et al. (1987). Retention data are given as the equivalent chain length (ECL) relative to the retention times for straight-chain saturated acetates. Electroantennography (EAG) and Linked Gas Chromatography-Electroantennography (GC-EAG). Male C. cephalonica moths were prepared for
FEMALECorcyra cephalonica PHEROMONE
1577
EAG recording as described by Beevor et al. (1986), except that the glass microelectrodes were inserted into the interstitial membrane between annuli of the intact antennal flagellum. The recording electrode was inserted into the distal end of one antenna, and the reference electrode into the proximal end of the other. The microelectrodes were filled with saline (Roelofs and Comeau, 1971) with 0.2% w/v agar added in order to reduce evaporation and avoid crystallization of dissolved salts in the microelectrodes. Test samples were syringed onto the inner walls of a disposable Pasteur pipet and blown over the antennal preparation with a pulse of nitrogen, as described by Beevor et al. (1986). Typically, an interval of 5 min was allowed between successive stimuli for the preparation to recover fully. Linked GC-EAG analyses with packed and fused silica capillary GC columns were carried out as described by Moorhouse et al. (1969) and Beevor et al. (1986). The GC column effluent was split 1 : 1 between the EAG preparation and the flame ionization detector. The GC columns used were as described above. Microchemieal Reactions. For bromination, a dilute solution of bromine in carbon tetrachloride (5/A) was added to the sample dissolved in carbon tetrachloride (5/A), and the mixture analyzed after 20 min at room temperature. The carbon tetrachloride was purified by passage through neutral alumina. Reductions were carried out by adding a few grains of lithium aluminium hydride to the sample dissolved in dichloromethane (15 #l). Oxidations were carried out with a saturated solution of pyridinium chlorochromate in dichloromethane for 20 min at room temperature. The pyridinium chlorochromate was washed with ether before use, and the dichloromethane was passed through neutral alumina. In all microchemical reactions, blanks for assay by GC or EAG were prepared under identical conditions using pure solvent instead of the test solution. Mass Spectrometry (MS). Linked GC-MS analyses of natural and synthetic materials were carried out in EI mode at 70 eV with a VG Micromass 7070F instrument and VG 2000 data system. This was coupled via an all-glass jet separator to a Pye 104 gas chromatograph fitted with a glass column (2.5 m x 2 mm ID) packed with 15% Carbowax 20 M on Chromosorb W and operated isothermally at 200~ Synthesis. 5,9,13-Trimethyl-2-pentadecanol (compound A) was prepared by a sequence starting with three successive Julia reactions (Julia et al., 1960) as shown in Figure 1. The " o n e - p o t " modification (Biernacki and Gdula, 1979) was used in these reactions. The yields became progressively poorer, but intermediates were available from a preparation of 4,8-dimethyldecanal, the aggregation pheromone of Tribolium spp. (Breuer et al., 1982). The yields are quoted for products purified by chromatography if necessary and distillation. 6,10,14-Trimethyl-2-pentadecanol (compound B) was prepared from far-
1578
~ .Br v
HALL ET AL.
{i) > 71%
~
~
(i) 9 1.7%
Br
OH
(iv)
~
95%
CHO
~
(vi) > 72%
v
(i)(ii)(iii) 22%
Br
~
v
~
/
~
V L v ~ j J V
.
OH
9
(v) 68%
~ OH
FIG. 1. Synthesis of 5,9,13-trimethyl-2-pentadecanol (A). Reagents: (i) Mg/Et20; cyclopropylmethyl ketone; H2SO4; (ii) KOAc/CH3CN/Adogen 464; (iii) K2CO3/MeOH; (iv) H2/PtO2/EtOH; (v) pyridinium chlorochromate/CH2C12;(vi) CH3MgI/Et20. Yields are for chromatographically homogeneous products after distillation.
nesol by standard reactions involving two-carbon homologation with ethyl acetoacetate, as shown in Figure 2. Bioassays. The olfactometric bioassay used was that described by Zagatti et al. (1987) and consisted of a glass tube olfactometer divided into seven sections. The positions of moths released into the olfactometer were recorded every minute for 5 min, and the attraction index (Dr) calculated as described by Zagatti et al. (1987). In order to determine the range of action of the female pheromone, the responses of individual male moths to female extract (1 female equivalent, FE) were recorded. In all, 99 male moths were observed in the olfactometer for 3-min periods, and the time and direction of their first shift were recorded along with their initial position. As controls, 28 males were observed under the same
FARNESOL
(iii) ( i v ) 25%
(i) 96%
~
9
OH
0
(ii) 9 93%
~
B
r OH
(v) 75% (a)
FIG. 2. Synthesis of 6,10,14-trimethyl-2-pentadecano!(B). Reagents: (i) H2/PtO2/EtOH; (ii) CBrg/PPh3/CH2C12; (iii) ethyl acetoacetate/Na/EtOH; (iv) 150~ (v) NaBH4/EtOH. Yields are for chromatographically homogeneous products after distillation.
FEMALECorcyra cephalonica PHEROMONE
1579
conditions with hexane (5 /zl) on the applicator, and there was no significant difference between the numbers of moths moving upwind and downwind after 3 min (X 2 = 3.7 with 4 df). The percentages of moths making an initial upwind shift after periods of 30 sec, 1 min 30 sec, and 3 rain were calculated for different initial positions, and these were compared with the corresponding results in the control tests using a 2 x 2 X 2 analysis. The precise role of the female-emitted pheromone in mating behavior was investigated using the synthetic pheromone. A solution of the pheromone (100 ng) in hexane (1 #1) was deposited onto the inner walls of a disposable Pasteur pipet, and air was blown at 200 ml/min through the pipet and over a pair of male moths caged in a small cylindrical box (5 x 8 cm diameter). A total of 15 pairs of males was tested with the pheromone and 15 pairs with hexane (1/zl) as controls. The results were recorded as the percentages of moths showing the following types of behavior within a 2-min observation period (Table 3): (1) calling posture (stationary, fanning wings); (2) search for females (walking, fanning wings); and (3) copulation attempts (moths circling and trying to mount one another). The test and control results were totally different, obviating the need for statistical analysis. RESULTS
Evidence for Female-Emitted Sex Pheromone. During initial studies of pheromone-mediated behavior in C. cephalonica, it was shown that live virgin female moths can attract male moths (Zagatti et al., 1987). A crude hexane extract of female abdominal tips (2.5 FE) was also shown to be attractive (Figure 3), demonstrating the olfactory nature of this phenomenon. In these tests, it seemed that male moths close to the pheromone source were markedly more stimulated than those further downwind. By recording the responses of individual male moths at different distances from the pheromone source it was shown that the percentage of moths responding to female extract (1 FE) by an upwind shift decreased sharply at distances greater than 13 cm from the source (Figure 4). At distances greater than 33 cm from the source, there was no significant difference between the responses in the presence or absence of the pheromone. Pheromone Identification. Whole-body washes or ovipositor washings from virgin female C. cephalonica moths caused EAG responses from the male moth, and linked GC-EAG analyses suggested that a single EAG-active component was present. Amounts of this component obtained under a variety of conditions were much less than 1 ng/female, and purification of these solvent washes by GC or liquid chromatography did not remove large amounts of impurities which prevented further analytical work.
1580
HALL ET AL.
attraction toward
of m a l e s
female
extract
40 w.
i
20 .-" /r
.,.O- .....
control
-'--*O
-O'"
0
-lO,
1
2 TiME
3
4
5
-MINUTES-
FIG. 3. Attraction indices for male C. cephalonica moths to female abdominal tip extract (2.5 FE; 68 males) and hexane (5/zl; 64 males) as control (eight replicates). Statistical analysis by Mann-Whitney test: difference between treatment and control significant at *P = 0.05; **P = 0.01.
Aeration of virgin female moths and collection of the volatiles emitted on Porapak Q gave significant amounts of pheromone that could be purified by liquid chromatography on Florisil. Assay of the fractions by EAG showed that essentially all the activity was eluted with 20 % ether in pentane. Thus the crude Porapak extract (0.5/~1 from 5 ml) gave an EAG response greater than 0.8 mV; the two active fractions (0.5 ~1 from 10 ml) each also gave an EAG response greater than 0.8 mV with no other fractions giving responses greater than 0.29 mV. Linked GC-EAG analysis of the crude Porapak extract on a GC column packed with Carbowax 20 M showed only one EAG-active component at ECL 15.36 (total response 1.0 mV; no other response greater than 0.15 mV). Analysis of the active fractions from liquid chromatography similarly showed one active component at the same retention time (total response 1.9 mV; background response 0.20 mV), and these fractions were sufficiently pure for carrying out microchemical reactions and GC-MS analysis on the active component. Approximately 5 ~g of this purified material was obtained after seven weeks of entrainment (1000-1500 moth days). When microgram quantities of synthetic long-chain hydrocarbons, aldehydes, acetates, and alcohols were chromatographed on Florisil under the same conditions, hydrocarbons were eluted with pentane, aldehydes with 2-5 % ether in pentane, acetates with 5 % ether in pentane, secondary alcohols with 10-20 % ether in pentane, and primary alcohols with 50% ether in pentane. The elution behavior of the active component was thus consistent with that of a long-chain, secondary alcohol.
FEMALECorcyra cephalonica PHEROMONE
1581
after 30 s. 100 o
~
after l m n 3 O s .
after 3 ran. "0
50 m
E
U O.
0 3-13
13-23
23-33
33-43
43-53
CONTRO L
Distance from the source (cm) FIG. 4. Responses of male C. cephalonica moths to female abdominal tip extract (1 FE; 99 males) and hexane (1 #1; 28 males) as control, relative to their initial distance from the source and the time after the start of the stimulation. The vertical axis represents the percentage of males which moved at least one section upwind in the olfactometer. Statistical analysis by X2 test: difference between treatment and control not significant, o; significant at *P = 0.05; **P = 0.01.
Bromination o f the crude Porapak extract caused no loss in E A G activity. GC analyses on a GC column packed with Carbowax 20 M showed that the purified active component was unaffected by bromination. Bromination o f standard synthetic compounds showed that unsaturated compounds and aldehydes reacted but saturated acetates, alcohols, and ketones were unchanged. On treatment o f the crude Porapak extract with lithium aluminium hydride in dichloromethane, E A G activity was lost, but the activity was completely restored when the reaction mixture was hydrolyzed with water. These results were consistent with the active component being a saturated alcohol. Reaction o f the purified active component with pyridinium chlorochromate
1582
HALL ET AL.
was monitored by GC analysis on columns packed with SE-30 and Carbowax 20 M. The active component at ECL 14.44 and 15.36 on the two columns reacted to give a product at ECL 14.30 and 14.33. These shifts in ECL on the two GC phases of - 0 . 1 4 and - 1 . 0 3 , respectively, were consistent with a methyl carbinol-to-methyl ketone conversion (Table 1). The mass spectrum of the active component (Figure 5a) showed ions of highest mass number at m/z 255 and 252, interpreted as loss of CH 3 and H20 from a saturated, 18-carbon alcohol of molecular weight 270. The remainder of the spectrum suggested an aliphatic rather than alicyclic structure. The GC retention times for the active component on fused silica capillary columns (Table 2) indicated that, if the component was a saturated 18-carbon alcohol with the alcohol in the 2 position, it must have several branches in the aliphatic chain. These branches would be favored fragmentation points in the mass spectrum (e.g., Carlson et al., 1978), and 5,9,13-trimethyl-2-pentadecanol (A) was prepared as a model compound. This compound had a mass spectrum (Figure 5c) significantly different from that of the active component and slightly longer GC retention times (Table 2). In view of the known shorter retention times of TABLE 1. RETENTION DATA FOR NATURAL AND SYNTHETIC ALCOHOLS AND CORRESPONDING CARBONYL COMPOUNDS ON PACKED G C COLUMNS a
ECL Compound Natural pheromone component Natural pheromone component + pyridinium chlorochromate (difference) 1 -Heptadecanol Heptadecanal (difference) 2 -Heptadecanol 2-Heptadecanone (difference) 3-I-Ieptadecanol 3 -Heptadecanone (difference) 4-Heptadecanol 4-Heptadecanone (difference)
SE-30
Carbowax 20 M
14.44
15.36
14.30 (-0.14) 15.75 15.05 ( - 0.70) 15.04 14.88 ( - 0.16) 14.95 15.00 (+0.05) 15.00 14.70 (-0.30)
14.33 ( - 1.03) 17.70 15.30 ( - 2.40) 16.31 15.31 ( - 1.00) 16.00 15.00 ( - 1.00) 15.86 14,43 ( - 1.43)
~Glass columns (1.8 m • 2 mm ID) packed with 2.5% SE-30 + 0.25% Carbowax 20 M or 1.5% Carbowax 20 M on 100-120 mesh Chromosorb G AW DMCS; temperature programmed from 120~ to 220~ at 4~ nitrogen carrier gas at 25 mllmin_ ECL = equivaJent chain tength.
FZMALECorcyra cephalonica PHEROMONE
1583
100" (o) Corcyro cepholonico pheromone 97
111 126
50
140
i
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I
1
167 I II
9
100
|
-
182 196 d [ 210 224 II I, ~ 200 i
-
9
9
|
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i
t
-
i
51
(b) ~
b
(B)
u) 0J 5o
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~g
83 I
a:
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,
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,
I
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100
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182 196 210
Icl v [ . / ~ v v [ ~
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. .iJ, I. I I, 1111;31~1~~ '
1;o
.
.
.
.
.
I 260
'
'
"
F[G. 5. Electron impact mass spectra of (a) EAG-active component in volatiles collected
from virgin female C. cephalonica moths; (b) synthetic 6,10,14-trimethyl-2-pentadecanol (B); (c) synthetic 5,9,13-trimethyl-2-pentadecanol (A).
1584
HALL ET AL.
TABLE 2. G C DATA FOR NATURAL AND SYNTHETIC COMPOUNDS ON FUSED SILICA CAPILLARY COLUMNS a
CP Sil 5 CB
CP Wax 57CB ECL
Natural pheromone component and 6,10,14-Trimethyl-2-pentadecanol (B) 5,9,13-Trimethyl-2-pentadecanol (A) 2-Octadecanol
14.42 14.52 16.02
15.50 15.57 17.45
Peak width at half height (sec) National pheromone component 6,10,14-Trimethyl-2-pentadecanol
3.4 9.0
8.0 11.4
~Columns (25 m x 0.32 m m ID) coated with CP Sil 5 CB (chemically bonded methylsilicone) or CP Wax 57CB (chemically bonded Carbowax 20 M); oven temperature 70~ for 2 min, then programmed at 20~ to 100~ then at 2~ to 200~ helium carrier gas at 0.4 kg/cm 2
iso-branched fatty acid methyl esters relative to the anteiso- isomers (e.g., Body, 1984), 6,10,14-trimethyl-2-pentadecanol (B) was then prepared. This compound had a mass spectrum (Figure 5b) that was essentially identical with that of the active component, and identical retention times on fused silica capillary GC columns (Table 2). The latter were confirmed by cochromatography of the natural and synthetic materials. The GC peak for the synthetic mixture of four diastereoisomers was broadened relative to that for the natural component on both columns (Table 2), suggesting that the natural component consisted of only one or a few of the possible diasteroisomers. Compound B elicited a significant EAG response from male C. cephalonica moths, while compound A showed no activity. Responses from the same moth to 3.5 ng of material delivered through the GC-EAG link were 2.31, 1.23, and 0.0 mV for the natural pheromone component, compound B, and compound A, respectively. The lower EAG response to the synthetic mixture of diastereoisomers and enantiomers also suggests that the natural material consists of only one or a few of the possible isomers. Bioassays of Synthetic Pheromone. Both candidate compounds A and B were tested as attractants in the olfactometer. At the 200 ng level, compound B was highly attractive to the male moths whereas A was inactive even at the 400 ng level (Figure 6a). The attraction indices for 200 ng of B were considerably higher than those evoked by 2.5 FE of female abdominal tip extract (Figure 3), and the tests were repeated with lower amounts of the synthetic material (Figure 6b). Male moths were significantly attracted to 2 ng of compound B.
FEMALECorcyra cephalonica PHEROMONE
a
~
B
:
1585
2
O
O
n
g
t.
"40 uJ
O Z ][
o2c p(J 4K Ir kI,1(
*** .O"
1
........
o .......
~
A: 400 ng
....
2
3
TIME
(MINUTES)
b
control
4
5
~
200 ng
,..40
?
z
o
2 ng
2C
1r
......
1 -10-
..-'"'~"
2
3
TIME
(MINUTES)
..........
4
control
5
Fie. 6. (a) Attraction indices for male C. cephalonica moths to compound A (400 ng; 67 males), compound B (200 ng; 67 males), and hexane (5 ~1; 64 males) as control (eight replicates). (b) Attraction indices for male C. cephalonica moths to compound B (20 ng, 30 males; 2 ng, 27 males) (four replicates). Statistical analyses by Mann-Whitney test: difference between treatment and control not significant, ns; significant at *P = 0.05; **P = 0.01; ***P = 0.001. The synthetic compound B was used to investigate further the effect of the female pheromone on male behavior. Table 3 shows the results of exposing pairs of virgin males in small cages to compound B. Before the test, 50 % o f the males were essentially stationary and fanning their wings. The male pheromone is emitted during wing-fanning (Zagatti et al., 1987), and it is assumed
1586
HALL ET AL.
TABLE 3. EFFECTS OF SYNTHETIC PHEROMONE ON BEHAVIOR OF MALE
C. cephalonica
Observed behavior (%)~ Before stimulation
After stimulation
Stimulant
Calling posture
Search for female
Calling posture
Search for female
Copulation atteml~tS
100 ng (B) 1 /zl hexane
50 57
0 0
0 47
87 0
53 0
a2 • 15 pairs of male moths observed within a 2-min period.
that this is "calling" behavior. In less than 2 min after stimulation with compound B, 87 % of the moths tested started to walk sinuously while continuing their wing-fanning. This behavior seems to indicate searching for a mate, and homosexual copulation attempts were observed in 53% of the pairs. During control tests with pure solvent, the initial percentage of calling males decreased slightly, possibly due to the air disturbance. Neither searching for a mate nor copulation attempts were observed. In the same experiment run with a dead female moth replacing one of the pair of live males, no copulation attempts were observed, showing the importance of visual and/or auditory cues in the courtship behavior.
DISCUSSION
Virgin female C. cephalonica moths have been shown to produce a pheromone that acts as a short-range attractant for the m a l e moths. Volatiles collected from virgin female moths contain one component that elicits EAG responses from male moths, and the available chemical, chromatographic, and spectroscopic data on this component are consistent with that of synthetic 6,10,14-trimethyl-2-pentadecanol (B). In support of this identification, compound B causes an EAG response from male C. cephalonica moths and is highly active in the bioassays. Furthermore, the closely related compound, 5,9,13trimethyl-2-pentadecanol (A) is biologically inactive. No attempt has been made to determine which of the four diastereoisomeric pairs of enantiomers of B is (are) produced by the female moth, but there is chemical and biological evidence that only one or a few of the isomers is involved. Thus the GC peak width for the natural pheromone component is narrower than that for the synthetic mixture of isomers, and the natural phero-
FEMALECorcyra cephalonica PHEROMONE
1587
mone causes a greater EAG response than that to an equal amount of the synthetic. It is also possible that other pheromone components are present, although these must be present in much lower amounts and/or cause a smaller EAG response than the major component. When this work was carried out, 6,10,14-trimethyl-2-pentadecanol (B) had not previously been found in an insect secretion. However, Burger et al. (1985) recently reported this compound to be a major component of the material produced in the male abdominal brushes of Eldana saccharina, another galleriid closely related to C. cephalonica. No biological function was reported. In most lepidopterous species, the female produces a long-range attractant pheromone. It has also been demonstrated in many of these species that the male produces a short-range courtship pheromone. Butler (1967) used the general term "aphrodisiac" for these, but this is inadequate to describe the many roles they may play (Boppr6, 1984). Short-range attraction of female Grapholitha molesta (Lepidoptera: Tortricidae) by the male was demonstrated by Baker et al. (1981), and the pheromone emitted from the male abdominal brushes was fully identified by Nishida et al. (1982). The bioassay was carried out over a distance of 2 cm. Ephestia elutella (Lepidoptera: Pyralidae; Phycitinae) is more closely related to C. cephalonica. The female produces a long-range attractant, and the males have been shown to produce a pheromone from glands in the forewings that causes the female moth to stop calling, flex the abdomen downwards, and turn (Krasnoff and Vick, 1984). This pheromone was characterized chemically by Phelan et al. (1986) and shown to contain several 3,-lactones and (E)-phytol [(E)-3,7,11,15-tetramethyl-2-hexadecen-l-ol], a compound not unrelated to the 6,10,14-trimethyl-2-pentadecanol (B) found to be produced by female C. cephalonica moths. By contrast, in the Galleriinae it seems that the long-range pheromone is typically produced by the male moths, evoking a searching behavior in the females (Zagatti et al., 1987, and references therein). This necessitates a cue from the female for the male to change behavior from passive calling to courtship directed towards the female. In Eldana saccharina it was shown that this cue is auditory in nature (Zagatti, 1981). In C. cephalonica the female moths emit a courtship pheromone that acts over a short distance to attract the male and cause him to attempt copulation. The site of production of the female pheromone has not been established, and histological examination has failed to show any glandular structures on the female abdomen. In the females of most Lepidoptera, the pheromone-producing glands are modifications of the intersegmental membranes between abdominal segments VIII and IX (Percy and Weatherston, 1974). In C. cephalonica it is possible that secretory cells are disseminated along the abdomen. Thus analyses
1588
HALL ET AL.
o f f e m a l e c u t i c u l a r w a s h e s s u g g e s t e d that m o r e p h e r o m o n e was obtained w h e n larger portions o f the a b d o m e n w e r e taken, and c o l l e c t i o n o f volatiles from f e m a l e m o t h s was f o u n d to be a better s o u r c e o f p h e r o m o n e than cuticular washing. It is uncertain h o w useful the synthetic p h e r o m o n e s will be in m o n i t o r i n g or control o f C. cephalonica. T h e f e m a l e - e m i t t e d p h e r o m o n e is probably too short-range in action to b e used alone. T h e m a l e - e m i t t e d p h e r o m o n e could be used to bait traps o r to disrupt m a t i n g if it causes a directed, a n e m o t a c t i c response in the f e m a l e m o t h , rather than a searching response as is the case with E. s a c c h a r i n a . W o r k to investigate this further is in progress. Acknowledgments--Thanks are due to Dr. Rick Hodges and other members of the TDRI Storage Department, Slough, England, for providing insect cultures, and to Miss Janet Robinson of TDRI for providing GC-MS data.
REFERENCES BAKER, T.C., NISHIDA, R. and ROELOFS,W.L. 1981. Close-range attraction of female Oriental fruit moth to herbal scent of male hairpencils. Science 214:1359-1361. BEEVOR, P:S., CORK, A., HALL, D.R., NESBITT,B.F., DAY, R.K., and MUMFORD,I.D. 1986. Components of female sex pheromone of cocoa pod borer moth, Conopomorpha cramerella. J. Chem. Ecol. 12:1-23. BIERNACKI,W., and GDULA, A. 1979. Modification of the method of Julia for the preparation of homoallylic bromides and iodides. Synthesis 1979:37-38. BODY, D.R. 1984. Branched-chain fatty acids, pp. 241-275, in H.K. Mangold (ed.). CRC Handbook of Chromatography. Lipids, Vol. 1. CRC Press, Boca Raton, Florida. BOPPRt~, M. 1984. Chemically mediated interactions between butterflies, pp. 259-275, in R.I. Vane-Wright and P.R. Ackery (eds.). The Biology of Butterflies. Academic Press, London. BREUER, E., DEUTSCH, J., and LAZAROVICI,P. 1982. A practical synthesis of the aggregation pheromone of Tribolium castaneum and Tribolium confusum. Chem. Ind. 1982:907-908. BURGER, B.V., MACKENROTH,W.M., SMITH, D., SPIES, H.S.C., and ATKINSON, P.R. 1985. Chemical composition of the wing gland and abdominal hair pencil secretions of the male African sugarcane borer, Eldana saccharina (Lepidoptera: Pyralidae). Z. Naturforsch. 40c:847-850. BUTLER,C.G. 1967. Insect pheromones. Biol. Rev. Cambridge Phil. Soc. 42:42-67. CARLSON,D.A., LANGLEY,P.A., and HUVTON,P. 1978. Sex pheromone of the tsetse fly: isolation, identification, and synthesis of contact aphrodisiacs. Science 201:750-753. JULIA, M., JULIA, S., and GUEGAN,R. 1960. Prrparation de composrs terprniques et apparentrs, ~tpartir de mrthyl cyclopropyl crtone. Bull. Soc. Chim. Fr. 1960:1072-1079. KRASNOFF,S.B., and VICK, K.W. 1984. Male wing-gland pheromone ofEphestia elutella. J. Chem. Ecol. 10:667-679. MOORHOUSE,I.E., YEADON,R., BEEVOR,P.S., and NESBITT,B.F. 1969. Method for use in studies of insect chemical communication. Nature 223:1174-1175. NISHIDA.R., BAKER,T.C., and ROELOFS,W.L. 1982. Hairpencil pheromone components of male Oriental fruit moths, Grapholitha molesta. J. Chem. Ecol. 8:947-959.
FEMALE Corcyra cephalonica PHEROMONE
1589
PERCY, J.E., and WHEATHERSTON,J. 1974. Gland structure and pheromone production in insects, pp. 11-34, in M.C. Birch (ed.). Frontiers of Biology, Vol. 32: Pheromones. North-Holland Publishing, Amsterdam. PHELAN, P.L., SinK, P.J., NORTHCOTT, C.J., TAN, S.H., and BAKER,T.C. 1986. Chemical identification and behavioral characterization of male wing pheromone of Ephestia elutella (Pyralidae). J. Chem. Ecol. 12:135-146. ROELOFS,W.L., and COMEAU,A. 1971. Sex pheromone perception: Electroantennogram responses of the red-banded leafrotler moth. J. Insect Physiol. 17:1969-1982. ZAGATTI,P. 1981. Comportement sexuel de la Pyrale de la canne ~ sucre: Eldana saccharina (Wlk.) 1i6 h deux ph6romones 6raises par le mgde. Behaviour 78:81-98. ZAGATTI, P., KUNESCH, G., RAMIANDRASOA,F., MALOSSE, C., HALL, D.R., LESTER, R., and NESBITT, B.F. 1987. Sex pheromones of rice moth, Corcyra cephalonica Stainton. I. Identification of male pheromone. J. Chem. Ecol. 13:1561-1573.
SEX PHEROMONES OF RICE MOTH, Corcyra cephalonica STAINTON II. Identification and Role of Female Pheromone
D.R. HALL, 1 A. CORK, 1 R. LESTER, l B.F. N E S B I T T , ~ and P. Z A G A T T I 2 ITropical Development and Research Institute 56/62 Gray's Inn Road, London WC1X 8LU, England 21NRA-CNRS, Laboratoire des Mddiateurs Chimiques Dnmaine de Brou~ssy, Magny-les-Hameaux F-78470 St-R~my-lks-Chevreuse, France (Received June 13, 1986; accepted September 29, 1986) Abstract--Laboratory investigations of mating behavior in the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae; Galleriinae) showed that male moths are attracted at short range to live, virgin female moths and to female abdominal-tip extract. Volatiles collected from virgin female moths contained one component eliciting an electroantennographic (EAG) response from the male moth, and the chemical, spectroscopic, and chromatographic data on this component were consistent with that of synthetic 6,10,14-trimethyl-2-pentadecanol. This compound caused an EAG response from the male moth and attracted male moths in the bioassay. The pheromone is thought to play a role in courtship, and the synthetic material was shown to cause the male moths to search for a mate and attempt copulation. Key Words--Rice moth, Corcyra cephalonica, Lepidoptera, Pyralidae, Galleriinae, female pheromone, courtship pheromone, olfactometer, electroantenogram, 6,10,14-trimethyl-2-pentadecanol.
INTRODUCTION Studies o f m a t i n g b e h a v i o r in the rice m o t h , Corcyra cephalonica (Lepidoptera: Pyralidae; Galleriinae) h a v e s h o w n that f e m a l e m o t h s are attracted to m a l e moths, and m a l e m o t h s are attracted to f e m a l e m o t h s (Zagatti et al., 1987). The m a l e m o t h s w e r e f o u n d to p r o d u c e ( E , E ) - and ( Z , E ) - f a r n e s a l (3,7,11-trim e t h y l - 2 , 6 , 1 0 - d o d e c a t r i e n a l ) f r o m glands in the f o r e w i n g s , and the synthetic 1575 0098-0331/8710700-1575505.00/0 9 1987 Plenum Publishing Corporation
1576
HALL ET AL.
compounds were shown to attract walking female moths (Zagatti et al., 1987). This paper describes identification of the female-produced pheromone and studies to establish its role in the mating behavior of this species. METHODS AND MATERIALS
Insect Material. Laboratory cultures of C. cephalonica were maintained as described by Zagatti et al. (1987). The identification work in London was carfled out with moths from cultures of Malawi origin. Moths from cultures originating in India were used for the bioassay work in France. Pheromone Collection and Purification. For bioassay work, the abdominal tips (three posterior segments including ovipositor) of 38 virgin female moths were excised 2 hr into the scotophase. The tips were extracted in 1 ml hexane for 2 hr and the extract filtered through glass wool and reduced to 100 # under nitrogen. For the identification work, ovipositor or whole-body washes from virgin female moths were made under a variety of conditions: age 0-7days; at various times throughout a 12 hr-12 hr light-dark cycle or under constant low light; in heptan~, ether, or dichloromethane. To collect pheromone by entrainment, 20-30 virgin female moths were placed in a silanized, 3-liter, bolt-head flask with two paper tissues. Laboratory air was drawn at 1 liter/min into the bottom of the flask through a charcoal filter (12 x 2 cm; 10-18 mesh) and out through two glass filters (1.5 cm ID) packed with 2.5 g Porapak Q (50-80 mesh) purified by washing well with dichloromethane. Pheromone was collected continuously under natural lighting conditions at 20-25~ Twice per week, the Porapak filters were extracted with a total of 25 ml dichloromethane, and dead moths were replaced with freshly emerged ones. After seven weeks of entrainment, the Porapak filter extracts were combined and evaporated to 5 ml on a rotary evaporator at or below room temperature. Florisil (0.5 g, 100-200 mesh) was added and the remaining solvent removed on the rotary evaporator. The coated Florisil was deposited on the top of a column of 9.5 g Florisil (30 x 1 cm) made up in pentane. This was then eluted with 50-ml aliquots of diethyl ether-pentane mixtures containing 0, 2, 5, 10, 20, 30, 50, 75, and 100% ether. Fractions of 10 ml were collected. Gas Chromatography (GC). Columns packed with SE-30 or Carbowax 20 M and fused silica capillary columns coated with CPSil 5 CB or CP Wax 57CB were used as described in Zagatti et al. (1987). Retention data are given as the equivalent chain length (ECL) relative to the retention times for straight-chain saturated acetates. Electroantennography (EAG) and Linked Gas Chromatography-Electroantennography (GC-EAG). Male C. cephalonica moths were prepared for
FEMALECorcyra cephalonica PHEROMONE
1577
EAG recording as described by Beevor et al. (1986), except that the glass microelectrodes were inserted into the interstitial membrane between annuli of the intact antennal flagellum. The recording electrode was inserted into the distal end of one antenna, and the reference electrode into the proximal end of the other. The microelectrodes were filled with saline (Roelofs and Comeau, 1971) with 0.2% w/v agar added in order to reduce evaporation and avoid crystallization of dissolved salts in the microelectrodes. Test samples were syringed onto the inner walls of a disposable Pasteur pipet and blown over the antennal preparation with a pulse of nitrogen, as described by Beevor et al. (1986). Typically, an interval of 5 min was allowed between successive stimuli for the preparation to recover fully. Linked GC-EAG analyses with packed and fused silica capillary GC columns were carried out as described by Moorhouse et al. (1969) and Beevor et al. (1986). The GC column effluent was split 1 : 1 between the EAG preparation and the flame ionization detector. The GC columns used were as described above. Microchemieal Reactions. For bromination, a dilute solution of bromine in carbon tetrachloride (5/A) was added to the sample dissolved in carbon tetrachloride (5/A), and the mixture analyzed after 20 min at room temperature. The carbon tetrachloride was purified by passage through neutral alumina. Reductions were carried out by adding a few grains of lithium aluminium hydride to the sample dissolved in dichloromethane (15 #l). Oxidations were carried out with a saturated solution of pyridinium chlorochromate in dichloromethane for 20 min at room temperature. The pyridinium chlorochromate was washed with ether before use, and the dichloromethane was passed through neutral alumina. In all microchemical reactions, blanks for assay by GC or EAG were prepared under identical conditions using pure solvent instead of the test solution. Mass Spectrometry (MS). Linked GC-MS analyses of natural and synthetic materials were carried out in EI mode at 70 eV with a VG Micromass 7070F instrument and VG 2000 data system. This was coupled via an all-glass jet separator to a Pye 104 gas chromatograph fitted with a glass column (2.5 m x 2 mm ID) packed with 15% Carbowax 20 M on Chromosorb W and operated isothermally at 200~ Synthesis. 5,9,13-Trimethyl-2-pentadecanol (compound A) was prepared by a sequence starting with three successive Julia reactions (Julia et al., 1960) as shown in Figure 1. The " o n e - p o t " modification (Biernacki and Gdula, 1979) was used in these reactions. The yields became progressively poorer, but intermediates were available from a preparation of 4,8-dimethyldecanal, the aggregation pheromone of Tribolium spp. (Breuer et al., 1982). The yields are quoted for products purified by chromatography if necessary and distillation. 6,10,14-Trimethyl-2-pentadecanol (compound B) was prepared from far-
1578
~ .Br v
HALL ET AL.
{i) > 71%
~
~
(i) 9 1.7%
Br
OH
(iv)
~
95%
CHO
~
(vi) > 72%
v
(i)(ii)(iii) 22%
Br
~
v
~
/
~
V L v ~ j J V
.
OH
9
(v) 68%
~ OH
FIG. 1. Synthesis of 5,9,13-trimethyl-2-pentadecanol (A). Reagents: (i) Mg/Et20; cyclopropylmethyl ketone; H2SO4; (ii) KOAc/CH3CN/Adogen 464; (iii) K2CO3/MeOH; (iv) H2/PtO2/EtOH; (v) pyridinium chlorochromate/CH2C12;(vi) CH3MgI/Et20. Yields are for chromatographically homogeneous products after distillation.
nesol by standard reactions involving two-carbon homologation with ethyl acetoacetate, as shown in Figure 2. Bioassays. The olfactometric bioassay used was that described by Zagatti et al. (1987) and consisted of a glass tube olfactometer divided into seven sections. The positions of moths released into the olfactometer were recorded every minute for 5 min, and the attraction index (Dr) calculated as described by Zagatti et al. (1987). In order to determine the range of action of the female pheromone, the responses of individual male moths to female extract (1 female equivalent, FE) were recorded. In all, 99 male moths were observed in the olfactometer for 3-min periods, and the time and direction of their first shift were recorded along with their initial position. As controls, 28 males were observed under the same
FARNESOL
(iii) ( i v ) 25%
(i) 96%
~
9
OH
0
(ii) 9 93%
~
B
r OH
(v) 75% (a)
FIG. 2. Synthesis of 6,10,14-trimethyl-2-pentadecano!(B). Reagents: (i) H2/PtO2/EtOH; (ii) CBrg/PPh3/CH2C12; (iii) ethyl acetoacetate/Na/EtOH; (iv) 150~ (v) NaBH4/EtOH. Yields are for chromatographically homogeneous products after distillation.
FEMALECorcyra cephalonica PHEROMONE
1579
conditions with hexane (5 /zl) on the applicator, and there was no significant difference between the numbers of moths moving upwind and downwind after 3 min (X 2 = 3.7 with 4 df). The percentages of moths making an initial upwind shift after periods of 30 sec, 1 min 30 sec, and 3 rain were calculated for different initial positions, and these were compared with the corresponding results in the control tests using a 2 x 2 X 2 analysis. The precise role of the female-emitted pheromone in mating behavior was investigated using the synthetic pheromone. A solution of the pheromone (100 ng) in hexane (1 #1) was deposited onto the inner walls of a disposable Pasteur pipet, and air was blown at 200 ml/min through the pipet and over a pair of male moths caged in a small cylindrical box (5 x 8 cm diameter). A total of 15 pairs of males was tested with the pheromone and 15 pairs with hexane (1/zl) as controls. The results were recorded as the percentages of moths showing the following types of behavior within a 2-min observation period (Table 3): (1) calling posture (stationary, fanning wings); (2) search for females (walking, fanning wings); and (3) copulation attempts (moths circling and trying to mount one another). The test and control results were totally different, obviating the need for statistical analysis. RESULTS
Evidence for Female-Emitted Sex Pheromone. During initial studies of pheromone-mediated behavior in C. cephalonica, it was shown that live virgin female moths can attract male moths (Zagatti et al., 1987). A crude hexane extract of female abdominal tips (2.5 FE) was also shown to be attractive (Figure 3), demonstrating the olfactory nature of this phenomenon. In these tests, it seemed that male moths close to the pheromone source were markedly more stimulated than those further downwind. By recording the responses of individual male moths at different distances from the pheromone source it was shown that the percentage of moths responding to female extract (1 FE) by an upwind shift decreased sharply at distances greater than 13 cm from the source (Figure 4). At distances greater than 33 cm from the source, there was no significant difference between the responses in the presence or absence of the pheromone. Pheromone Identification. Whole-body washes or ovipositor washings from virgin female C. cephalonica moths caused EAG responses from the male moth, and linked GC-EAG analyses suggested that a single EAG-active component was present. Amounts of this component obtained under a variety of conditions were much less than 1 ng/female, and purification of these solvent washes by GC or liquid chromatography did not remove large amounts of impurities which prevented further analytical work.
1580
HALL ET AL.
attraction toward
of m a l e s
female
extract
40 w.
i
20 .-" /r
.,.O- .....
control
-'--*O
-O'"
0
-lO,
1
2 TiME
3
4
5
-MINUTES-
FIG. 3. Attraction indices for male C. cephalonica moths to female abdominal tip extract (2.5 FE; 68 males) and hexane (5/zl; 64 males) as control (eight replicates). Statistical analysis by Mann-Whitney test: difference between treatment and control significant at *P = 0.05; **P = 0.01.
Aeration of virgin female moths and collection of the volatiles emitted on Porapak Q gave significant amounts of pheromone that could be purified by liquid chromatography on Florisil. Assay of the fractions by EAG showed that essentially all the activity was eluted with 20 % ether in pentane. Thus the crude Porapak extract (0.5/~1 from 5 ml) gave an EAG response greater than 0.8 mV; the two active fractions (0.5 ~1 from 10 ml) each also gave an EAG response greater than 0.8 mV with no other fractions giving responses greater than 0.29 mV. Linked GC-EAG analysis of the crude Porapak extract on a GC column packed with Carbowax 20 M showed only one EAG-active component at ECL 15.36 (total response 1.0 mV; no other response greater than 0.15 mV). Analysis of the active fractions from liquid chromatography similarly showed one active component at the same retention time (total response 1.9 mV; background response 0.20 mV), and these fractions were sufficiently pure for carrying out microchemical reactions and GC-MS analysis on the active component. Approximately 5 ~g of this purified material was obtained after seven weeks of entrainment (1000-1500 moth days). When microgram quantities of synthetic long-chain hydrocarbons, aldehydes, acetates, and alcohols were chromatographed on Florisil under the same conditions, hydrocarbons were eluted with pentane, aldehydes with 2-5 % ether in pentane, acetates with 5 % ether in pentane, secondary alcohols with 10-20 % ether in pentane, and primary alcohols with 50% ether in pentane. The elution behavior of the active component was thus consistent with that of a long-chain, secondary alcohol.
FEMALECorcyra cephalonica PHEROMONE
1581
after 30 s. 100 o
~
after l m n 3 O s .
after 3 ran. "0
50 m
E
U O.
0 3-13
13-23
23-33
33-43
43-53
CONTRO L
Distance from the source (cm) FIG. 4. Responses of male C. cephalonica moths to female abdominal tip extract (1 FE; 99 males) and hexane (1 #1; 28 males) as control, relative to their initial distance from the source and the time after the start of the stimulation. The vertical axis represents the percentage of males which moved at least one section upwind in the olfactometer. Statistical analysis by X2 test: difference between treatment and control not significant, o; significant at *P = 0.05; **P = 0.01.
Bromination o f the crude Porapak extract caused no loss in E A G activity. GC analyses on a GC column packed with Carbowax 20 M showed that the purified active component was unaffected by bromination. Bromination o f standard synthetic compounds showed that unsaturated compounds and aldehydes reacted but saturated acetates, alcohols, and ketones were unchanged. On treatment o f the crude Porapak extract with lithium aluminium hydride in dichloromethane, E A G activity was lost, but the activity was completely restored when the reaction mixture was hydrolyzed with water. These results were consistent with the active component being a saturated alcohol. Reaction o f the purified active component with pyridinium chlorochromate
1582
HALL ET AL.
was monitored by GC analysis on columns packed with SE-30 and Carbowax 20 M. The active component at ECL 14.44 and 15.36 on the two columns reacted to give a product at ECL 14.30 and 14.33. These shifts in ECL on the two GC phases of - 0 . 1 4 and - 1 . 0 3 , respectively, were consistent with a methyl carbinol-to-methyl ketone conversion (Table 1). The mass spectrum of the active component (Figure 5a) showed ions of highest mass number at m/z 255 and 252, interpreted as loss of CH 3 and H20 from a saturated, 18-carbon alcohol of molecular weight 270. The remainder of the spectrum suggested an aliphatic rather than alicyclic structure. The GC retention times for the active component on fused silica capillary columns (Table 2) indicated that, if the component was a saturated 18-carbon alcohol with the alcohol in the 2 position, it must have several branches in the aliphatic chain. These branches would be favored fragmentation points in the mass spectrum (e.g., Carlson et al., 1978), and 5,9,13-trimethyl-2-pentadecanol (A) was prepared as a model compound. This compound had a mass spectrum (Figure 5c) significantly different from that of the active component and slightly longer GC retention times (Table 2). In view of the known shorter retention times of TABLE 1. RETENTION DATA FOR NATURAL AND SYNTHETIC ALCOHOLS AND CORRESPONDING CARBONYL COMPOUNDS ON PACKED G C COLUMNS a
ECL Compound Natural pheromone component Natural pheromone component + pyridinium chlorochromate (difference) 1 -Heptadecanol Heptadecanal (difference) 2 -Heptadecanol 2-Heptadecanone (difference) 3-I-Ieptadecanol 3 -Heptadecanone (difference) 4-Heptadecanol 4-Heptadecanone (difference)
SE-30
Carbowax 20 M
14.44
15.36
14.30 (-0.14) 15.75 15.05 ( - 0.70) 15.04 14.88 ( - 0.16) 14.95 15.00 (+0.05) 15.00 14.70 (-0.30)
14.33 ( - 1.03) 17.70 15.30 ( - 2.40) 16.31 15.31 ( - 1.00) 16.00 15.00 ( - 1.00) 15.86 14,43 ( - 1.43)
~Glass columns (1.8 m • 2 mm ID) packed with 2.5% SE-30 + 0.25% Carbowax 20 M or 1.5% Carbowax 20 M on 100-120 mesh Chromosorb G AW DMCS; temperature programmed from 120~ to 220~ at 4~ nitrogen carrier gas at 25 mllmin_ ECL = equivaJent chain tength.
FZMALECorcyra cephalonica PHEROMONE
1583
100" (o) Corcyro cepholonico pheromone 97
111 126
50
140
i
10(?-
I
1
167 I II
9
100
|
-
182 196 d [ 210 224 II I, ~ 200 i
-
9
9
|
2i2 -
i
t
-
i
51
(b) ~
b
(B)
u) 0J 5o
97
~g
83 I
a:
111 126 167 .I
,
In.
[i
. I,
~
252 ,
,
I
2OO
100
loot s7 71 83
182 196 210
Icl v [ . / ~ v v [ ~
cAI
I
I
~$2
. .iJ, I. I I, 1111;31~1~~ '
1;o
.
.
.
.
.
I 260
'
'
"
F[G. 5. Electron impact mass spectra of (a) EAG-active component in volatiles collected
from virgin female C. cephalonica moths; (b) synthetic 6,10,14-trimethyl-2-pentadecanol (B); (c) synthetic 5,9,13-trimethyl-2-pentadecanol (A).
1584
HALL ET AL.
TABLE 2. G C DATA FOR NATURAL AND SYNTHETIC COMPOUNDS ON FUSED SILICA CAPILLARY COLUMNS a
CP Sil 5 CB
CP Wax 57CB ECL
Natural pheromone component and 6,10,14-Trimethyl-2-pentadecanol (B) 5,9,13-Trimethyl-2-pentadecanol (A) 2-Octadecanol
14.42 14.52 16.02
15.50 15.57 17.45
Peak width at half height (sec) National pheromone component 6,10,14-Trimethyl-2-pentadecanol
3.4 9.0
8.0 11.4
~Columns (25 m x 0.32 m m ID) coated with CP Sil 5 CB (chemically bonded methylsilicone) or CP Wax 57CB (chemically bonded Carbowax 20 M); oven temperature 70~ for 2 min, then programmed at 20~ to 100~ then at 2~ to 200~ helium carrier gas at 0.4 kg/cm 2
iso-branched fatty acid methyl esters relative to the anteiso- isomers (e.g., Body, 1984), 6,10,14-trimethyl-2-pentadecanol (B) was then prepared. This compound had a mass spectrum (Figure 5b) that was essentially identical with that of the active component, and identical retention times on fused silica capillary GC columns (Table 2). The latter were confirmed by cochromatography of the natural and synthetic materials. The GC peak for the synthetic mixture of four diastereoisomers was broadened relative to that for the natural component on both columns (Table 2), suggesting that the natural component consisted of only one or a few of the possible diasteroisomers. Compound B elicited a significant EAG response from male C. cephalonica moths, while compound A showed no activity. Responses from the same moth to 3.5 ng of material delivered through the GC-EAG link were 2.31, 1.23, and 0.0 mV for the natural pheromone component, compound B, and compound A, respectively. The lower EAG response to the synthetic mixture of diastereoisomers and enantiomers also suggests that the natural material consists of only one or a few of the possible isomers. Bioassays of Synthetic Pheromone. Both candidate compounds A and B were tested as attractants in the olfactometer. At the 200 ng level, compound B was highly attractive to the male moths whereas A was inactive even at the 400 ng level (Figure 6a). The attraction indices for 200 ng of B were considerably higher than those evoked by 2.5 FE of female abdominal tip extract (Figure 3), and the tests were repeated with lower amounts of the synthetic material (Figure 6b). Male moths were significantly attracted to 2 ng of compound B.
FEMALECorcyra cephalonica PHEROMONE
a
~
B
:
1585
2
O
O
n
g
t.
"40 uJ
O Z ][
o2c p(J 4K Ir kI,1(
*** .O"
1
........
o .......
~
A: 400 ng
....
2
3
TIME
(MINUTES)
b
control
4
5
~
200 ng
,..40
?
z
o
2 ng
2C
1r
......
1 -10-
..-'"'~"
2
3
TIME
(MINUTES)
..........
4
control
5
Fie. 6. (a) Attraction indices for male C. cephalonica moths to compound A (400 ng; 67 males), compound B (200 ng; 67 males), and hexane (5 ~1; 64 males) as control (eight replicates). (b) Attraction indices for male C. cephalonica moths to compound B (20 ng, 30 males; 2 ng, 27 males) (four replicates). Statistical analyses by Mann-Whitney test: difference between treatment and control not significant, ns; significant at *P = 0.05; **P = 0.01; ***P = 0.001. The synthetic compound B was used to investigate further the effect of the female pheromone on male behavior. Table 3 shows the results of exposing pairs of virgin males in small cages to compound B. Before the test, 50 % o f the males were essentially stationary and fanning their wings. The male pheromone is emitted during wing-fanning (Zagatti et al., 1987), and it is assumed
1586
HALL ET AL.
TABLE 3. EFFECTS OF SYNTHETIC PHEROMONE ON BEHAVIOR OF MALE
C. cephalonica
Observed behavior (%)~ Before stimulation
After stimulation
Stimulant
Calling posture
Search for female
Calling posture
Search for female
Copulation atteml~tS
100 ng (B) 1 /zl hexane
50 57
0 0
0 47
87 0
53 0
a2 • 15 pairs of male moths observed within a 2-min period.
that this is "calling" behavior. In less than 2 min after stimulation with compound B, 87 % of the moths tested started to walk sinuously while continuing their wing-fanning. This behavior seems to indicate searching for a mate, and homosexual copulation attempts were observed in 53% of the pairs. During control tests with pure solvent, the initial percentage of calling males decreased slightly, possibly due to the air disturbance. Neither searching for a mate nor copulation attempts were observed. In the same experiment run with a dead female moth replacing one of the pair of live males, no copulation attempts were observed, showing the importance of visual and/or auditory cues in the courtship behavior.
DISCUSSION
Virgin female C. cephalonica moths have been shown to produce a pheromone that acts as a short-range attractant for the m a l e moths. Volatiles collected from virgin female moths contain one component that elicits EAG responses from male moths, and the available chemical, chromatographic, and spectroscopic data on this component are consistent with that of synthetic 6,10,14-trimethyl-2-pentadecanol (B). In support of this identification, compound B causes an EAG response from male C. cephalonica moths and is highly active in the bioassays. Furthermore, the closely related compound, 5,9,13trimethyl-2-pentadecanol (A) is biologically inactive. No attempt has been made to determine which of the four diastereoisomeric pairs of enantiomers of B is (are) produced by the female moth, but there is chemical and biological evidence that only one or a few of the isomers is involved. Thus the GC peak width for the natural pheromone component is narrower than that for the synthetic mixture of isomers, and the natural phero-
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mone causes a greater EAG response than that to an equal amount of the synthetic. It is also possible that other pheromone components are present, although these must be present in much lower amounts and/or cause a smaller EAG response than the major component. When this work was carried out, 6,10,14-trimethyl-2-pentadecanol (B) had not previously been found in an insect secretion. However, Burger et al. (1985) recently reported this compound to be a major component of the material produced in the male abdominal brushes of Eldana saccharina, another galleriid closely related to C. cephalonica. No biological function was reported. In most lepidopterous species, the female produces a long-range attractant pheromone. It has also been demonstrated in many of these species that the male produces a short-range courtship pheromone. Butler (1967) used the general term "aphrodisiac" for these, but this is inadequate to describe the many roles they may play (Boppr6, 1984). Short-range attraction of female Grapholitha molesta (Lepidoptera: Tortricidae) by the male was demonstrated by Baker et al. (1981), and the pheromone emitted from the male abdominal brushes was fully identified by Nishida et al. (1982). The bioassay was carried out over a distance of 2 cm. Ephestia elutella (Lepidoptera: Pyralidae; Phycitinae) is more closely related to C. cephalonica. The female produces a long-range attractant, and the males have been shown to produce a pheromone from glands in the forewings that causes the female moth to stop calling, flex the abdomen downwards, and turn (Krasnoff and Vick, 1984). This pheromone was characterized chemically by Phelan et al. (1986) and shown to contain several 3,-lactones and (E)-phytol [(E)-3,7,11,15-tetramethyl-2-hexadecen-l-ol], a compound not unrelated to the 6,10,14-trimethyl-2-pentadecanol (B) found to be produced by female C. cephalonica moths. By contrast, in the Galleriinae it seems that the long-range pheromone is typically produced by the male moths, evoking a searching behavior in the females (Zagatti et al., 1987, and references therein). This necessitates a cue from the female for the male to change behavior from passive calling to courtship directed towards the female. In Eldana saccharina it was shown that this cue is auditory in nature (Zagatti, 1981). In C. cephalonica the female moths emit a courtship pheromone that acts over a short distance to attract the male and cause him to attempt copulation. The site of production of the female pheromone has not been established, and histological examination has failed to show any glandular structures on the female abdomen. In the females of most Lepidoptera, the pheromone-producing glands are modifications of the intersegmental membranes between abdominal segments VIII and IX (Percy and Weatherston, 1974). In C. cephalonica it is possible that secretory cells are disseminated along the abdomen. Thus analyses
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o f f e m a l e c u t i c u l a r w a s h e s s u g g e s t e d that m o r e p h e r o m o n e was obtained w h e n larger portions o f the a b d o m e n w e r e taken, and c o l l e c t i o n o f volatiles from f e m a l e m o t h s was f o u n d to be a better s o u r c e o f p h e r o m o n e than cuticular washing. It is uncertain h o w useful the synthetic p h e r o m o n e s will be in m o n i t o r i n g or control o f C. cephalonica. T h e f e m a l e - e m i t t e d p h e r o m o n e is probably too short-range in action to b e used alone. T h e m a l e - e m i t t e d p h e r o m o n e could be used to bait traps o r to disrupt m a t i n g if it causes a directed, a n e m o t a c t i c response in the f e m a l e m o t h , rather than a searching response as is the case with E. s a c c h a r i n a . W o r k to investigate this further is in progress. Acknowledgments--Thanks are due to Dr. Rick Hodges and other members of the TDRI Storage Department, Slough, England, for providing insect cultures, and to Miss Janet Robinson of TDRI for providing GC-MS data.
REFERENCES BAKER, T.C., NISHIDA, R. and ROELOFS,W.L. 1981. Close-range attraction of female Oriental fruit moth to herbal scent of male hairpencils. Science 214:1359-1361. BEEVOR, P:S., CORK, A., HALL, D.R., NESBITT,B.F., DAY, R.K., and MUMFORD,I.D. 1986. Components of female sex pheromone of cocoa pod borer moth, Conopomorpha cramerella. J. Chem. Ecol. 12:1-23. BIERNACKI,W., and GDULA, A. 1979. Modification of the method of Julia for the preparation of homoallylic bromides and iodides. Synthesis 1979:37-38. BODY, D.R. 1984. Branched-chain fatty acids, pp. 241-275, in H.K. Mangold (ed.). CRC Handbook of Chromatography. Lipids, Vol. 1. CRC Press, Boca Raton, Florida. BOPPRt~, M. 1984. Chemically mediated interactions between butterflies, pp. 259-275, in R.I. Vane-Wright and P.R. Ackery (eds.). The Biology of Butterflies. Academic Press, London. BREUER, E., DEUTSCH, J., and LAZAROVICI,P. 1982. A practical synthesis of the aggregation pheromone of Tribolium castaneum and Tribolium confusum. Chem. Ind. 1982:907-908. BURGER, B.V., MACKENROTH,W.M., SMITH, D., SPIES, H.S.C., and ATKINSON, P.R. 1985. Chemical composition of the wing gland and abdominal hair pencil secretions of the male African sugarcane borer, Eldana saccharina (Lepidoptera: Pyralidae). Z. Naturforsch. 40c:847-850. BUTLER,C.G. 1967. Insect pheromones. Biol. Rev. Cambridge Phil. Soc. 42:42-67. CARLSON,D.A., LANGLEY,P.A., and HUVTON,P. 1978. Sex pheromone of the tsetse fly: isolation, identification, and synthesis of contact aphrodisiacs. Science 201:750-753. JULIA, M., JULIA, S., and GUEGAN,R. 1960. Prrparation de composrs terprniques et apparentrs, ~tpartir de mrthyl cyclopropyl crtone. Bull. Soc. Chim. Fr. 1960:1072-1079. KRASNOFF,S.B., and VICK, K.W. 1984. Male wing-gland pheromone ofEphestia elutella. J. Chem. Ecol. 10:667-679. MOORHOUSE,I.E., YEADON,R., BEEVOR,P.S., and NESBITT,B.F. 1969. Method for use in studies of insect chemical communication. Nature 223:1174-1175. NISHIDA.R., BAKER,T.C., and ROELOFS,W.L. 1982. Hairpencil pheromone components of male Oriental fruit moths, Grapholitha molesta. J. Chem. Ecol. 8:947-959.
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PERCY, J.E., and WHEATHERSTON,J. 1974. Gland structure and pheromone production in insects, pp. 11-34, in M.C. Birch (ed.). Frontiers of Biology, Vol. 32: Pheromones. North-Holland Publishing, Amsterdam. PHELAN, P.L., SinK, P.J., NORTHCOTT, C.J., TAN, S.H., and BAKER,T.C. 1986. Chemical identification and behavioral characterization of male wing pheromone of Ephestia elutella (Pyralidae). J. Chem. Ecol. 12:135-146. ROELOFS,W.L., and COMEAU,A. 1971. Sex pheromone perception: Electroantennogram responses of the red-banded leafrotler moth. J. Insect Physiol. 17:1969-1982. ZAGATTI,P. 1981. Comportement sexuel de la Pyrale de la canne ~ sucre: Eldana saccharina (Wlk.) 1i6 h deux ph6romones 6raises par le mgde. Behaviour 78:81-98. ZAGATTI, P., KUNESCH, G., RAMIANDRASOA,F., MALOSSE, C., HALL, D.R., LESTER, R., and NESBITT, B.F. 1987. Sex pheromones of rice moth, Corcyra cephalonica Stainton. I. Identification of male pheromone. J. Chem. Ecol. 13:1561-1573.
