Journal of Chemical Ecology, Vol. 15, No. 6, 1989
ISOLATION A N D I D E N T I F I C A T I O N OF M A L E M E D F L Y A T T R A C T I V E C O M P O N E N T S IN Litchi chinensis STEMS A N D Ficus SPP. STEM E X U D A T E S 1
J.D.
WARTHEN,
JR., 2 and D.O.
McINNIS 3
2Insect Chemical Ecology Laboratory USDA, ARS, PSI Beltsville, Maryland 20705 3Tropical Fruit and Vegetable Research Laboratory USDA, ARS Honolulu, Hawaii 96804 (Received May 24, 1988; accepted September 26, 1988)
Abstract--Short-range attraction/feeding stimulation of male Mediterranean fruit flies [Ceratitis capitata (Wiedemann), (Diptera: Tephritidae)] to a stem extract of a host plant, Litchi chinensis Sonn. (Sapindaceae), and to milky exudates from stems of nonhost plants, Ficus retusa L. and F. benjamina L. (Moraceae), were attributed to the presence of the sesquiterpene a-copaene. The presence of a-copaene in the milky exudate from stems of F. benghalensis L. is also suggested as eliciting similar behavioral responses in male medflies. The presence of minor quantities of c~-ylangene in the plants and its contributory effects to the behavioral response of male medflies is discussed. Short-range attraction/feeding stimulation of male medflies to equal amounts of ~-ylangene-free c~-copaene samples (94.5% +), prepared from ~-copaene-enriched angelica seed oil and copaiba oil, respectively, showed no difference in intensity of response, a-Ylangene elicited a slightly less intense response for male medflies than c~-copaene. Key Words--Litchi chinensis, Sapindaceae, Ficus retusa, Ficus benjamina, Ficus benghalensis, Moraceae, Ceratitis capitata, Diptera, Tephritidae, Mediterranean fruit fly, medfly, ~-copaene, a-ylangene. Names of products in this paper are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA.
1931
1932
WARTHEN AND MCINNIS INTRODUCTION
Naturally occurring attractants for Ceratitis capitata (Wiedemann) have been found in extracts and oils of many plants (Beroza and Green, 1963a). These include extracts of Alpinia officinarum, Anaphalia margaritacea, Angelica spp.,
Berberis vulgaris, Chimaphila umbellata, Conioselinum chinense, Dendrobium superbum, Equisetum arvense, Fabriana imbricata, Festuca spp., Heracleum lanatum, Juglans nigra, Lycopersicon esculentum, Rhamnus frangula, Rosa centifolia, Tilia europaea, and oils (Teranishi et al., 1987) of Abies alba, angelica seed (Steiner et al., 1957; Beroza and Green, 1963b; Fomasiero et al., 1969; Jacobson et al., 1987), copaiba, cubeb, cyste, gingergrass, grapefruit, hop, lemon, orange, Canadian pine needle, Pinus pumilio, P. sylvestris, sweet orange, orange peel, and ylang-ylang. The present study extends this list of naturally occurring attractants for C. capitata to the host plant, Litchi chinensis Sonn. (Sapindaceae) (Back and Pemberton, 1918a,b), Ficus benghalensis L. (Moraceae)/F. indica, which has conflicting reports of host vs. nonhost classification (Quayle, 1938; Back and Pemberton, 1918a,b), and several nonhost Ficus spp. L. chinensis, litchi or lychee, is a medium sized-tree that is native to the humid regions of southern China, where it has grown for several thousand years. Thousands of tons of the dried fruit are exported for use by Chinese people living abroad. These fruits are known as Chinese litchi nuts in the Western world. The trees are also cultivated in the American subtropics (Dahlgren, t947). McInnis and Warthen (1988) observed that the exudate from a stem of F. benjamina L., weeping fig, attracted male medflies and stimulated them to feed. Other species of Ficus, namely F. benghalensis L. (Indian banyan) and F. retusa L. (Indian laurel fig), produced the same behavioral responses (Table 1). We did not investigate F. carica, the common fig, which is a host for medfly. This plant is listed as being heavily or generally infested by medflies (Back and Pemberton, 1918a,b). A detailed procedure was developed to isolate and identify the short-range attractants/feeding stimulants from L. chinensis; this same procedure was also applied to the Ficus spp. for isolation and identification.
METHODS AND MATERIALS
L. chinensis stems were collected and dried at two locations: the Tropical Agriculture Research Station, Mayaguez, Puerto Rico, and at the Subtropical Horticulture Research Laboratory, Miami, Florida. Leaves, small stems, and twigs of F. retusa and F. benjamina were also obtained and dried at Mayaguez.
1933
MEDFLY ATTRACTANTS
T A B L E | . SHORT-RANGE LABORATORY MEDFLY ATTRACTANCY/FEEDING STIMULATION SCORING SYSTEMa
Qualitative score Negative ( - ) Slight to negative (sl to - ) Slight (sl) +
++ +++
Quantitative scoreb No reaction above normal "passerby" activity. 0-5 flies feeding or "sitting" on blotter within 5 min of start. 5-10 flies feeding, "mating, ''C or resting on blotter within 5 min. 10-20 flies feeding, etc., on blotter, within 2 min. 20-40 flies feeding, etc., on blotter within 2 rain. Male "mating" activity heavy. 40-60 flies feeding, etc., within 2 min. Male "mating" activity heavy.
a McInnis and Warthen, 1988. bApproximately 200 flies/sex for each test. c "Mating" by males consisted of attempted copulations by two or more individuals mounted in a row.
Leaf stem exudates were obtained from F. benjamina at the U S D A Tropical Fruit and Vegetable Laboratory, Honolulu, Hawaii; from F. retusa on the east side of Oahu, Hawaii; and from F. benghalensis at the U.S. Botanic Gardens, Washington, D.C. A small sample of F. benghalensis fresh leaves was also obtained from the latter source. Solvents and Sesquiterpenes. n-Hexane and isooctane were H P L C grade; all other solvents were reagent grade. The sources o f sesquiterpenes, including enriched a - c o p a e n e samples from angelica seed oil and copaiba root oil, are listed in Table 2. Extractions and Essential Oils. Dried stems (231.3 g) of Puerto Rican L. chinensis were ground in a W i l e y mill and extracted with n-hexane for 24 hr in a Soxhlet apparatus; the marc was extracted with ethyl ether for 24 hr. Similar extractions o f the dried stems o f Miami L. chinensis were also performed. Fresh leaves (91.0 g) o f F. benghalensis were ground in a Waring blender with n-hexane and extracted for 18 hr with n-hexane in a Soxhlet apparatus; the marc was extracted with ethyl ether for 18 hr. Dried leaves, small stems, and twigs (307.3 g) o f F. retusa were ground in a W i l e y mill and extracted with ethyl ether for 72 hr in a Soxhlet apparatus. Similar extractions o f the dried leaves, small stems, and twigs (323.9 g) o f F . benjamina were also performed. A steam distillate (collected in n-hexane, utilizing a continuous extraction head for 24 hr) was prepared from each o f the following: 64.3 g ethyl ether extract o f Puerto Rican L. chinensis (1498 g), 26.1 g ethyl ether extract o f Miami L. chinensis (804 g), 48.9 g ethyl ether extract o f F . retusa (307.3 g), and 40.6 g ethyl ether extract o f F. benjamina (323.9 g).
1934
WARTHEN AND MCINNIS
TABLE 2. GLC AND HPLC RETENTION TIMES OF STANDARD SESQUITERPENES
Sesquiterpene
Source
GLC Retention timea (min)
HPLC Retention timeb (min)
( +)-a-Longipinene ( + ) -Longicyclene c~-Ylangene ~-Copaene ( -)-Isolongifolene ( +)-Longifolene a-Cedrene ( - ) -/3-Caryophyllene (-)-Thujopsene (+)-Calarene (/3-Gurjunene) ( +)-Aromadendrene c~-Humulene (a-Caryophyllene) Alloaromadendrene ~-Gurjunene Eremophilene (labeled as valencene)
Fluka Fluka c a Fluka Aldrich Aldrich Fluka Fluka Fluka
8.79 9.03 9.16 9.24 9.29 9.64 9.76 9.92 10.06 10.13
20.93
Fluka Fluka
10.29 10.59
Fluka Fluka Roth
10.69 11.07 11.07
27.06 29.33 22.06 12.60 e
32.60 e 13.46
aRetention times are for elution comparison within the table and are not exactly superimposable upon retention times in Figure 2. bSome tailing of the monounsaturated sesquiterpenes occurs with these analyses. cObtained from Professor V. Herout, Institute of Organic Chemistry and Biochemistry, Czeckoslovak Academy of Science, Prague. aObtained from Professor Buehi, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts. Also obtained from M. Jaeobson; USDA-Retired, Collaborator (Jacobson et al., 1987). e(-)-Thujopsene and (-)-/3-caryophyllene were separated on 3 /~m silica under the same HPLC conditions, giving 9.46 and 11.26 min retention times, respectively.
E a c h o f the a b o v e extracts w e r e dried (sodium sulfate) and concentrated in v a c u o . T h e steam distillates w e r e concentrated to 5.0 ml solutions. Exudates. E x u d a t e s o f F. benjamina and F. benghalensis w e r e collected with Pasteur pipets f r o m snapped l e a f stems. T h e exudates w e r e refrigerated as such or m i x e d with distilled w a t e r (2 drops e x u d a t e / 1 . 0 ml water) and refrigerated. T w o o f the latter F. benghalensis mixtures w e r e extracted with 1.0 ml n - h e x a n e and 1.0 m l ethyl acetate, respectively. L e a f stem exudates o f F. retusa w e r e similarly collected, and trunk exudates w e r e c o l l e c t e d through a w o u n d m a d e in the trunk. T h e s e exudates w e r e extracted w i t h an e q u i v a l e n t v o l u m e o f m e t h y l e n e chloride; the organic layer was stored u n d e r refrigeration. Open-Column Chromatography. M i l l i p o r e Waters A s s o c i a t e s S e p - P a k sil-
MEDFLYATTRACTANTS
1935
ica cartridges were prewashed with 5.0 ml methylene chloride and then 10.0 ml n-hexane. The n-hexane extract (1-5 drops from a Pasteur pipet) of L. chinensis was placed on the Sep-Pak and eluted with three 5.0-ml portions of n-hexane followed by 5.0 ml methylene chloride; four 5.0-ml fractions (A1-4, Scheme 1) were collected. To 437 g Bio-Rad Bio-Sil A (100-200 mesh) was added an aqueous, saturated solution of 109.25 g reagent-grade silver nitrate with shaking; the mixture was heated at 125~ overnight. The first two fractions, A1 and A2, were
Puerto Rican Litchi chinensis 231.3 g ground dried stems
I
I Soxhlet extraction, n-hexane, 24 hr
I
---2.0171 g (+++) + Marc
t
I I 0.87%
1.5125 g (++)
~ . Open-column Ag-silica
__J
t
5-2.8% =-copaene
A3 A4 B1 B2 B3 B4 B5-B9 BI0 BII BI2
Open-column Ag-silica
yield
Soxhlet extraction, ethyl ether, 24 hr
Silica Sep-Pak
(++) (++)-(+++) 48% ~-copaene (+++) 6-caryophyllene
Ci-4 75% =-copaene, 11% ~-ylangene (+)-(++)
HPLC
Ag-silica
I c7-14
I
~-copaene SCHEME 1. See Table 1 for qualitative scoring (+, + +, + + +) for short-range laboratory medfly attractant/feeding stimulation.
1936
WARTHZNAND MCINNIS
placed on a column (3.0 x 0.5 cm ID) containing 0.32 g of this adsorbent (20% silver nitrate) that was first wetted and prewashed with 2.0 ml n-hexane. Portions (0.7 ml each) of n-hexane, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 8.0, 16.0, 32.0, 64.0% ethyl ether/n-hexane, and ethyl ether were added to the column and twelve 0.7-ml fractions (BI-12) were collected. B3, concentrated to a 50-/~1 volume, was placed on another prewashed silver nitrate-Bio-Sil A column of the same configuration and eluted with portions (0.7 ml each) of n-hexane, 0.5, 1.0, 1.5, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10.0, 20.0, and 40.0% ethyl ether/n-hexane; each of the fourteen 0.7-ml fractions (C1-14) was concentrated. A 4.1-ml methylene chloride extract of 4.0 mt F. retusa exudate was concentrated under nitrogen to 0.6 ml; three 0.2-ml aliquots of this concentrate were each placed on a prewashed silica Sep-Pak and eluted with n-hexane as previously described. The first fractions from each Sep-Pak were combined and concentrated under nitrogen to 0.2 ml. An aliquot (150 #1) of this concentrate was placed on a 20 % silver nitrate-Bio-Sil A column as previously described and etuted with portions (0.7 ml each) of n-hexane, 1.0, 2.0, 4.0, 8.0, 12.0, 18.0, 27.0, 40.0, 60.0% ethyl ether/n-hexane, and ethyl ether. High-Performance Liquid Chromatography (HPLC). Analytical and preparative HPLC were carried out on a Waters Associates ALC-100 equipped with a model 720 system controller, a model 730 data module, two model 6000A pumps, a U6K injector, and a model 440 absorbance detector with an extended wavelength module at 214 rim. Silver nitrate (20 % w/w) on 3 /zm Hypersil (Shandon) was prepared by adding a solution of 2.5 g reagent grade silver nitrate in 70 ml acetonitrile to a mixture of 10 g 3/zm Hypersil in acetonitrile (Heath et al., 1975). The mixture was concentrated in vacuo to dryness, and 4.0 g was added to 30 ml carbon tetrachloride and sonicated for several minutes. The mixture was added to a stainless-steel reservoir and topped off with carbon tetrachloride (Heath et al., 1977). The mixture was pumped into a 30 x 0.39 cm ID stainless-steel column followed by 50 ml n-hexane at 9000 psi air pressure. Then, the packed column was flushed with 5% ethyl ether-isooctane at 1.5 ml/min, until 400 ml was collected, and further flushed with isooctane until all traces of ethyl ether were removed. Sesquiterpenes (50-100 /xg) in a volume of 5-100/zl n-hexane solution were injected for analysis at a flow rate of 0.5 ml/min isooctane. After maintaining this flow rate for 15 min, a linear flow program was then initiated over 5 rain to 1.0 ml/min isooctane followed by a linear gradient program over the next 10 min to 0.5% ethyl ether-isooctane. C5, containing about 12 /zg ~-copaene in a 78-/~1 volume of solvent, was also injected; c~-copaene was collected and concentrated under nitrogen to a 100-/~1 volume. GLC. A Hewlett-Packard 5880A gas chromatograph equipped with a cap-
MEDFLY ATTRACTANTS
1937
illary injector system and a flame ionization detector was used for GLC analyses. A Hewlett-Packard 12 m x 0.2 mm ID dimethyl silicone (DMS) fusedsilica capillary column was used for the analyses. Temperatures employed for GLC were: injection port 200~ detector 220~ DMS column, 50~ for 5 min, then programmed at 20~ to 105~ maintained at 105~ for 7.25 min, then programmed at 10~ to 125~ Total run time was 22 min. A split ratio of 1 : 188 was used with a helium flow of 2.2 cm3/min plus an appropriate amount of helium makeup gas. Standard sesquiterpene samples were dissolved in n-hexane to make 0.1% solutions, and 1 /zl of the solutions were injected for GLC analyses. The concentration of unknowns in solution for GLC analyses was adjusted to match that of standards in most cases. Gas Chromatography-Mass Spectrometry (GC-MS). Mass spectra were recorded on a Finnigan GC-MS 4500 with a 6000 data system. The GC component was equipped with a 60 m x 0.25 mm ID DB-1 capillary column (J&W Scientific) for sesquiterpene analyses in Figures 1A and 1B and a 50 m x 0.25 m m ID Ultra 1 (100% dimethyl polysiloxane gum) capillary column (HewlettPackard) for analyses of o~-copaene in Figures 1C and 1D. In the EI mode, 70 eV energy was used. Bioassay. A rapid and efficient short-range laboratory bioassay was developed (Mclnnis and Warthen, 1988) to evaluate crude and fractionated plant extracts, purified phytochemicals, and standard sesquiterpenes as attractants/ feeding stimulants for medflies, C. capitata. The bioassay's qualitative scoring system with a quantitative and behavioral basis appears in Table 1. Typically, 1-t~g samples of purified phytochemicals and standard sesquiterpenes were utilized. The concentrations of crude and fractionated plant extracts were adjusted according to the percentage of active substance present. RESULTS
A Soxhlet extraction (2.5-5.1% yield) of Puerto Rican L. chinensis (Scheme 1) was also obtained with ethyl ether for 72-96 hr (the extract obtained during the last 24 hr gave a rapid + + + in the bioassay); extraction over 96 hr
FIG. 1. Structural formulae of (-)-~-copaene (A) and (+)-a-ylangene (B).
1938
WARTHEN AND MCINNIS
with n-hexane alone gave only a 1.7% yield with a slow + + + in the bioassay. The Miami L. chinensis, when extracted with ethyl ether in a Soxhlet for 72 hr, gave a yield ranging from 2.6 to 5.9% that elicited less biological activity than the Puerto Rican variety. Biologically active A1 and A2 (Scheme 1) from the Puerto Rican L. chinensis n-hexane extract showed the possible presence of ~-ylangene (Figure 1B), ot-copaene (2.5-2.8%, Figure 1A), ~-caryophyllene, o~-humulene, longifolene, and eremophilene by retention time comparison with standard sesquiterpenes (Table 2) via GLC analyses. Chromatography of A1 and A2 revealed biological activity centered in B3, which appeared to contain a-copaene (48 %) by GLC analyses. The presence of/~-caryophyllene (95%) in B10 was confirmed by GC-MS with an NIH/EPA Spectral Library match. This substance was isolated and identified since it was listed as an insect attractant for medflies and Oriental fruit flies (Beroza and Green, 1963a). However, a Fluka ~-caryophyllene sample (99.7 % by GLC analysis) indicated no biological activity for medflies or Oriental fruit flies. K&K and Givaudan/~-caryophyllene samples that were attractive to male medflies revealed the presence of 0.38-0.57% and 0.41% ~-copaene, respectively, by GLC retention time comparison with standard ~-copaene. Chromatography of B3 revealed biological activity centered in C5, which appeared to contain 75 % ~-copaene and 11% ~-ylangene by GLC analyses. HPLC of C5 effectively separated the two closely related sesquiterpenes (Table 2) to give a + + + biologically active substance (95.6 % ~-copaene by capillary GLC analysis), whose mass spectral fragmentation matched that of c~-copaene in the NIH/EPA Mass Spectral Library. ~-Copaene samples of 94.5% and 94.8 % purity (capillary GLC analyses) prepared from o~-copaene-enriched samples isolated from angelica seed oil and copaiba oil, respectively, were prepared by this method. Each of these three 94.5 % + pure c~-copaene samples exhibited the same biological response of the same intensity for equal treatments. Due to the small observed optical rotation of cr and insufficient amounts of this sesquiterpene isolated from L. chinensis, it was not possible to obtain a specific optical rotation. Although there was not a sufficient quantity of the substance in C5 whose GLC retention time coincided with c~-ylangene for isolation, there was an adequate source of c~-ylangene in the ~-copaene-enriched samples from angelica seed oil and copaiba oil. c~-Ylangene, free of ~-copaene, was prepared by HPLC from these samples. This c~-ylangene, as well as standard a-ylangene (Table 2, 98.9 % pure by capillary GLC analysis), having identical GLC and HPLC retention times, initiated the same medfly biological response of the same intensity for equal treatments; this response (+ + to + + +) was slightly less than that (+ + +) for the c~-copaene for equal treatments (McInnis and Warthen, 1988).
MEDFLY ATTRACTANTS
1939
Steam distillates of Puerto Rican and Miami L. chinensis also contained 1.91-2.66% (Figure 2A) and 18.39-28.47% (Figure 2B) c~-copaene (retention times, 8.96 and 8.97 min, respectively), the structure of which was confirmed by GC-MS with an NIH/EPA Mass Spectral Library match for ~-copaene. The overall yields of a-copaene were 0.001% and 0.0003 %, respectively, agreeing with the difference in biological activity for the two samples. The Miami L. chinensis seemed to be devoid of a GLC peak coincident for a-ylangene, whereas the Puerto Rican L. chinensis steam distillate had a 0.25 % peak (coincident for a-ylangene) and a 2.01% ot-copaene peak, roughly 1 : 8. Crude leaf stem exudates (10-50 /zl) of F. benjamina and F. retusa (McInnis and Warthen, 1988), as well as F. benghalensis, were each shown to attract male medflies in the laboratory bioassay. Ethyl acetate and n-hexane extracts of fresh leaf stem exudates of F. benghalensis attracted male medflies, but solvent extracts of leaf stem exudate of F. benjamina that were processed after shipment from Hawaii showed less activity. Therefore, it was essential to extract the leaf stem exudates immediately upon harvest. Capillary GLC analyses of n-hexane, ethyl ether, ethanol, and acetone extracts of small quantities of F. benjamina and F. benghalensis leaf stem exudate did not reveal useful data due to the low concentration of terpenes. A larger quantity of exudate was then obtained from a large specimen of F. retusa; the leaf stem exudate was more attractive to medflies than the trunk exudate (McInnis and Warthen, 1988). The methylene chloride extracts of the fresh exudates were biologically active to medfly and maintained this activity over time. Open-column chromatography, via Sep-Pak (to remove latexlike materials) and 20% silver nitrate-Bio-Sil A, gave a fraction that showed medfly activity analogous to B3-B4 in Scheme 1 and that contained a real peak coincident with the retention time of standard c~-copaene by comparison with a blank open-column chromatographic run. To show the presence of c~-copaene in F. retusa (307.3 g) and F. benjamina (323.9 g) and by inference in their exudates, the steam distillates (48.9 g and 40.6 g of material, respectively) were analyzed by GLC. Chromatograms of the distillates (Figures 2C and 2D) indicated the presence of 21.0 % c~-copaene in F. retusa and 11.0% ot-copaene in F. benjamina (retention times, 8.95 and 8.96 min, respectively); structure was confirmed in each case by GC-MS with an NIH/EPA Spectral Library match for c~-copaene. If c~-ylangene were present in these distillates, it would be present at < 0.6%, based on coincident retention times with standard a-ylangene. The yield, based on dried plant specimens, of a-copaene was 1.146 mg (0.0004%) from F. retusa and 0.802 mg (0.0002%) from F. benjamina. Capillary GLC analysis did not reveal the presence of e~-copaene in extracts of a small specimen of fresh F. benghalensis leaves; in all probability, this was
~Rs
% C O N P E U S A % E D ~NRLYSIS
RT
A~EA
8.96
I
P-
e~-COPAENs
9.58 10.89 i8,21 I8.74 ! t~, 87 i1 36 lk.d7
8~s
o;g
1.55 :8.11 4.~9 5.18 4,17 17.92 4.;9~ 2.58
2,659 31.@18 6.999 8.871 7.136 38.69! 8.223 4.489
~'; CQMPENSI~[ED ~r~ALYSIS RT
~RE~
EIRE~, L
6.21 6,97 3,63
2.36 I . 77 1,17
0.4,4! 8. ~3~ 0,552
;9.78
2.29
8.97 G - C O P A E N E 7 3 . 8 S '
d
8RE~
!. E~B2 189~93
9 a9
:.Si
8.855
9,58 9, 79 18,@9 18,22 18,36
[email protected] ii,83 '-1 - Z 2 11.36 t L, 48 i!,74 !?,14
4Y~. 88 1.88 3.68 13.68 2.40 21,$6
2.!. 7 ~ 0, 7~4 i .739 6.473 !. !35
[email protected]
12.42 !3,59 !7,81
79
Z. 7-97
3 , :,T 4.-10 7 . ~_.3
'_. 472 29 ~, 5 9 i
26,5,~
!2.572
14.19 4.39 4.87 2.~i
6,7:4
2,u378 l 9 985 1.37a
g io
,o
FIo. 2. Capillary GLC of the essential oils from Puerto Rican Litchi chinensis dried stems (A); Miami L. chinensis dried stems (B); Ficus retusa dried leaves, small stems, and twigs (C); and F. benjamina dried leaves, small stems, and twigs (D).
=.-,.
]]MS~
16.48
12
ll.~.b
tQ_~?
0.2
9,57
~.ib
6.51
MM
-.1
c.l pa
8,SI
~o
N N
10,92
r -< o3
$m ~
0
iIo21 11.74
13.63
~~i.aa
8.77
12.
12
~.57
6.51
r o ~.J or, r_rl ~, o'. r_r~ , - . . ~
-.a r o ~:, ~
~c* c.J
."r
v~
F ,~
.-4
N
11.73
8.95
1942
WARTHEN AND MCINNIS
due to the size of the specimen extracted. An adequate amount of this species could not be located for extraction; but, it is very likely that the attractiveness of its leaf stem exudate is also due to the presence of c~-copaene.
DISCUSSION
Fornasiero et al. (1969) isolated c~-copaene and ot-ylangene (20:1) from angelica seed oil and demonstrated equal attractiveness to male medflies in lab bioassays (Guiotto et al., 1972); c~-pinene and ot-phellandrene from this oil were slightly attractive, and other sesquiterpenic fractions were more strongly attractive. The structure of a-copaene was resolved by de Mayo et al. (1965) and Kapadia et al. (1965), and the structure of ot-ylangene was resolved by Motl et al. (1965) and Ohta and Hirose (1969). c~-Copaene was first isolated from African copaiba oil (Kapadia et al., 1965; Wenninger et al., 1967a,b; Lawrence, 1980); it was also isolated from Cedrella toona (de Mayo et al., 1965; Kapadia et al., 1965), chloranthus oil (de Mayo et al., 1965), ylang-ylang oil (Wenninger et al., 1966, 1967b), peppermint oil (Wenninger et al., 1967b), grapefruit and orange oils (Hunter and Brogden, 1964; Veldhuis and Hunter, 1968; Teranishi et al., 1987), and cubeb oil (Ohta et al., 1968; Teranishi et al., 1987). More recent reports indicate its presence in Yucca gloriosa (Wang and Kameoka, 1977), guava (MacLeod and de Troconis, 1982), juniper berry and oil of sage (Formacek and Kubeczka, 1982), pineapple fruits (Berger et al., 1983), leaves of Siparuna guianensis (Aubl.) (Antonio et al., 1984), corn leaf volatiles (Buttery and Ling, 1984), wheat (Buttery et al., 1985), cotton (Elzen et al., 1985), Lippia nodiflora (L.) Greene (Elakovich and Stevens, 1985), and Heracleum dissectum Ledeb. (Papageorgiou et al., 1985). c~-Copaene usually exists as the levorotatory enantiomer; however, Jacobson et al. (1984) reported that oz-copaene from angelica seed oil is dextrorotatory (Jacobson et al., 1987). Teranishi et al., (1987) also reported that the c~-copaene from orange oil was dextrorotatory. c~-Ylangene has been isolated from Schizandra chinensis (Turcz) Baill by Motl et al. (1963) and Ohta et al. (1968), and traces have been reported in corn leaf volatiles (Buttery and Ling, 1984) and numerous other sources. Racemic c~-copaene was first synthesized via a 17-step route by Heathcock (1966) who, with Heathcock et al. (1967), reported the synthesis of racemic c~-ylangene. Subsequently, Corey and Watt (1973) reported simpler syntheses of racemic ~-copaene and c~-ylangene. The short-range attraction/feeding stimulation of male medflies to a host plant, L. chinensis, from Puerto Rico (Mclnnis and Warthen, 1988) was demonstrated and shown to be caused by 0.001% c~-copaene in the dried twigs. Smaller amounts of a substance with a GLC retention time coincident to that of
MEDFLY ATTRACTANTS
1943
c~-ylangene, in the steam distillate (Figure 2A), also probably contributes to the behavioral response of male medfties since the o~-ylangene standard (Table 2) elicits a similar response. The ratio of ~-copaene to c~-ylangene (retention times, 8.96 and - 8.83 min, respectively) in the steam distillate was 8 : 1. Through parallel bioassay/isolation of fractions from Litchi (Scheme 1), ~-copaene (95.6%, c~-ylangene-free) was isolated by HPLC as an active component. The behavioral response to this sample was identical to those produced by ~-copaene samples (~-ylangene-free), each with a purity of 94.5 % and 94.8 %, prepared by HPLC of ot-copaene-enriched samples from angelica seed oil and copaiba oil, respectively. No difference in behavioral response was noted to the purified ~-copaene from copaiba and the purified o~-copaene from angelica, each having opposing rotations, as reported by Jacobson et al. (1984, 1987); the implication is that optical rotation of these enantiomers is not relevant over short distances. ~-Ylangene standard (98.9%) and a-ylangene (a-copaene-free) samples, the latter being prepared from a-copaene-enriched samples from angelica seed oil or copaiba oil, gave the same male medfly response but slightly less intense than with o~-copaene. The presence of c~-ylangene in L. chinensis from Miami was not detectable, even though there was 18.4% c~-copaene (Figure 2B, retention time, 8.97 min) in the steam distillate as compared to 2.7% o~-copaene (Figure 2A, retention time, 8.96 min) in the Puerto Rican sample. However, the overall yield of c~-copaene was three times greater from the Puerto Rican sample than from the Miami sample. ~-Caryophyllene in L. chinensis (B3, Scheme 1) was ruled out as a medfly attractant through bioassay even though it had been reported as an attractant (Beroza and Green, 1963a). Old samples of ~-caryophyllene were probably attractive because of the presence of c~-copaene as an impurity. The short-range attraction/feeding stimulation of male medfiies to the milky exudate of a nonhost plant, F. retusa (Mclnnis and Warthen, 1988), was also shown to be caused by ~-copaene through a fractionation similar to Scheme 1. Fractions that would correspond to B3-4 showed a real GLC peak corresponding to ~-copaene; and a steam distillate of leaves, small stems, and twigs revealed the presence of 21.0% ~-copaene (Figure 2C, retention time, 8.95 min). A similar behavioral response to the milky exudate of the other nonhost plant, F. benjamina (Mclnnis and Warthen, 1988), was also probably due to ~-copaene since a similar steam distillate revealed the presence of 11.0% c~-copaene (Figure 2D, retention time, 8.96 min). If we had had a large enough sample of F. benghalensis to do a steam distillation, c~-copaene would probably have been revealed as the component in the milky exudate responsible for eliciting a similar behavioral response of male medflies. If c~-ylangene were present in F. retusa and F. benjamina, contributing to the medfly behavioral response, it would be present in the steam distillate (Figures 2C and 2D) at < 0.6% based on coincident retention times.
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T h e d i s c o v e r y o f s y n t h e t i c s o r p h y t o c h e m i c a l s as s h o r t - r a n g e a t t r a c t a n t s / f e e d i n g s t i m u l a n t s f o r t h e m a l e m e d f l y is a n i m p o r t a n t area o f r e s e a r c h . S u c h m a t e r i a l s m a y b e u s e f u l i n s t u d y i n g this s h o r t - r a n g e p h e n o m e n o n a n d m a y e v e n b e u s e d as a n e r a d i c a t i o n t o o l w h e n c o m b i n e d w i t h a b a i t - t o x i c a n t s y s t e m . Acknowledgments--We thank the following USDA, ARS employees: Dr. Franklin W. Martin, Tropical Agriculture Research Station, Mayaguez, Puerto Rico, and Paul Soderholm, Subtropical Horticulture Research, Miami, Florida, for samples of L. chinensis; Francisco Vazquez, Tropical Agricultural Research Station, Mayaguez, Puerto Rico, for samples of F. retusa and F. benjamina; and Dr. James A. Duke, Narcotics Laboratory, Beltsville, Maryland, for arranging for these samples. The authors also thank USDA, ARS, Beltsville, Maryland, employees E. David DeVilbiss and Dr. Barbara A. Leonhardt, Insect Chemical Ecology Laboratory, for GC-MS data, M. Jacobson (retired; collaborator) for enriched a-copaene samples from angelica seed oil and copaiba root oil, and Dr. Charles W. Woods, Insect Reproduction Laboratory, for packing HPLC columns. We also thank Richard R. Barrett and Rayma Overton, U.S. Botanic Gardens, Washington, D.C., for leaves and leaf stem exudate of F. benghalensis; Professor V. Herout, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Science, Prague, for an a-ylangene sample; and Professor G. Buchi, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, for an a-copaene sample.
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