Journal of Chemical Ecology, Vol. 26, No. 2, 2000

0098-0331/ 00/ 0200-0533$18.00/ 0 2000 Plenum Publishing Corporation

533

MUTUAL REPRODUCTIVE BENEFITS BETWEENA WILD ORCHID, Bulbophyllum patens, AND Bactrocera

FRUIT FLIES VIA A FLORAL SYNOMONE

KENG-HONG TAN1,* and RITSUO NISHIDA2

1School of Biological SciencesUniversiti Sains Malaysia11800 Penang, Malaysia

2Laboratory of Chemical Ecology, Graduate School of AgricultureKyoto University

Kyoto, 606-8502, Japan

(Received April 20, 1999; accepted October 11, 1999)

Abstract—The solitary flower of Bulbophyllum patens selectively attractsmale fruit flies of several Bactrocera species with a specific fragrance inthe rain forest of Malaysia. It temporarily traps flies between its hingedsee-saw lip and column for pollination. The attractant component is zingerone[4-(4-hydroxy-3-methoxyphenyl)-2-butanone], a pungent essence of ginger.Zingerone has a structure resembling two major fruit fly attractants (methyleugenol and raspberry ketone) and shows potency to attract a wide rangeof fruit fly species (B. carambolae, B. caudata, B. cucurbitae, B. tau, andB. umbrosa). A fruit fly visitor is rewarded by feeding on zingerone, andin return it removes the pollinarium and then transfers it to another flower.Males of the melon fly acquire the floral essence and selectively store it inthe pheromone gland to attract conspecific females. Males of B. papayae,however, convert zingerone to zingerol in the crop. The latter compound isstored in the rectal gland and subsequently released to attract females. Thisprovides a rare example of a mutualistic interaction between insects and plantsvia a floral synomone, in which both organisms obtain advantages directlybenefiting their reproductive systems.

Key Words—Bulbophyllum patens, Orchidaceae, fruit flies, Bactrocera,zingerone, attractant, pollination, pheromone, synomone, coevolution.

*To whom correspondence should be addressed.

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INTRODUCTION

In the course of coevolutionary interactions between plants and pollinators, flow-ers produce various kinds of colorful pigments and fragrances to attract a diver-sity of pollinators to the nectar. In complex vegetation, however, flowers tend toselect pollinators from several species of insects, so that their pollen can be deliv-ered efficiently to the flowers of the same species without letting the insects wan-der along the way. Some orchid flowers attract specific bee species by deceivingmales with an imitation signal of female odor and mimetic appearance (withoutrewards of sugars in nectar) (Whitten et al., 1989; Borg-Karlson, 1990). In neo-tropical orchid–euglossine bee associations, the males seek the specific orchid fra-grance to acquire volatile components (Dressler, 1982). In these instances, how-ever, the biological function of the flower fragrances in the pollinators’ life historyhas not been clearly elucidated. We show here a unique case of orchid–insect inter-action in which both the plant and the pollinators receive the reproductive advan-tage mediated by a synomonal substance produced by the flowers.

Bulbophyllum (Orchidaceae) is probably the largest orchid genus and con-tains about 1000 species (Vermeulen, 1991). Under this genus is a section ofSestochilus Benth & Hook. f., which contains 60–70 species. Among the knownspecies is Bu. patens King, an epiphytic orchid found in the lowland forests(0–100 m) of Borneo Island, Peninsular Malaysia, and Sumatra (Figure 1). Theflowers selectively attract male fruit flies of several Bactrocera species (Tephriti-dae), including destructive agricultural pest species, with a specific fragrance andtemporarily trap flies between a hinged see-saw lip and the column for pollina-tion (Tan, 1998). The Bactrocera species are strictly classified into two distinctgroups by the strong affinity of males to specific natural attractant chemicals,either methyl eugenol (ME, 1) or raspberry ketone (RK, 2) (Figure 2) (Drew,1974; Fletcher, 1987). One of us (Tan) further observed that the Bu. patensflower attracts both ME-sensitive species (B. carambolae Drew & Hancock, B.papayae Drew & Hancock, etc.) and RK-sensitive species [B. cucurbitae Coquil-lett, B. tau (Walker), etc.] in Malaysia. Hitherto, no compound has been knownto attract both groups. Thus, we initially assumed the flower contained both MEand RK. However, the flower was found to produce a single compound thatattracted both groups of the flies. We report the identification of the active prin-ciple and on the ecological significance of the compound for both the orchid andthe fruit flies.

METHODS AND MATERIALS

Observation of Fruit Flies Attracted to Bu. patens Flower. Individual fruitflies attracted to Bu. patens flowers were observed in the wild. Wherever pos-sible, the behavior of fly or flies on a flower was filmed with an 8-mm video

ORCHID SYNOMONE FOR FRUIT FLIES 535

FIG. 1. A male fruit fly (Bactrocera papayae) feeding on a lateral sepal of a wildorchid flower, Bulbophyllum patens. The fly bears a pollinarium from another B. patens(small arrow) on its thoracic dorsum and is attracted to the flower by a fragrantessence—zingerone (3), which the fly consumes. Note that the see-saw lip (broad arrow)is hinged to the base of the floral column of this nonresupinate orchid flower, whichcontains the highest concentration of zingerone.

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FIG. 2. Gas chromatogram of floral extract of a wild orchid, Bulbophyllum patens [24-m× 0.2-mm fused silica column coated with cross-linked methyl silicone HP-1, 0.33-mmfilm thickness, programmed from 808 (2 min holding) to 2408 at a rate of 108C/ min,FID detection] and structure of chemicals. 1: Methyl eugenol, 2: Raspberry ketone, 3:Zingerone, 4: Zingerol.

camcorder. Whenever possible, the attracted flies were collected after behavioralobservation. However, if the attracted fly was not a common Bactrocera species,then it was collected immediately for identification. To confirm floral attractionthrough fragrance, a day or two before blooming, a bud was enclosed in a blackwire-mesh cage (6 cm diam. × 9 cm long). Fly attraction was observed beforeand after the flower bloomed.

Fruit Fly Cultures. B. carambolae (reared on a semiartificial medium withcalrambola fruit), B. papayae, and B. cucurbitae (artificial medium) were cul-tured under ambient conditions in the insectarium. Virgin male and female adults

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were isolated two to three days after emergence and maintained in separate cages.They were provided with a yeast, protein hydrolysate, and sugar mixture andwater (fly food).

Instruments. For GC-MS of the volatile components, we used a HitachiM-80 mass spectrometer (electron impact, at 70 eV) connected to a GC column(24 m × 0.2 mm fused silica column coated with cross-linked methyl siliconeHP-1, 0.33-mm film thickness, with helium as carrier gas) programmed from808C (2 min holding) to 2408C at a rate of 108C/ min. The 1H NMR spectra wererecorded with a Bruker AC300 spectrometer (300 MHz). GC quantifications ofvolatile chemicals were done on an HP 5790A gas chromatograph with the samecapillary column and under the same program conditions as above by comparingthe FID intensities with those of the standard sample of known concentrationsby using a HP 3396 integrator (Hewlett Packard).

Extraction and Purification of Bu. patens Floral Fragrance. Flowers of Bu.patens were collected initially from Tenom Orchid Centre, Department of Agri-culture, Sabah, in 1995, and then from a forest in Perak, Peninsular Malaysiain 1996. Two flowers (collected on September 12, 1995 and April 21, 1996)were extracted with ethanol (1 ml × 3). The combined ethanolic solution wasfiltered (Advantec filter paper, No. 2) and concentrated under reduced pressure.The crude extract (16 mg) was partitioned between a 1 : 1 mixture of benzeneand ethyl acetate (2 ml × 2) and water (2 ml). The organic layer was driedover anhydrous sodium sulfate and evaporated in vacuo to give an oily mate-rial (6 mg) that was chromatographed on a silica gel column (0.4 g Wako gelC-200, Wako Pure Chemical Industries, Osaka). Fractions (10 ml) were elutedin sequence with mixtures of increasing concentration of methyl acetate in hex-ane (v/ v, ml) as follows: fractions 1 (0%, 4), 2 (3%, 2), 3 (10%, 2), 4 (15%, 2),5 (20%, 1), 6 (20%, 3), 7 (25%, 1), 8 (25%, 1), 9 (30%, 1) and 10 (30%, 3).The major active compound (zingerone) was purified from fraction 6 by high-performance liquid chromatography (HPLC) on a Sumipax column [Nucleosil100-5, 8 mm ID × 300 mm (packed by Sumitomo Chemical Industries, Tokyo].The solvent was 24% methyl acetate in hexane, 2.5 ml/ min.

Quantification of Zingerone in Floral Parts. Floral lip, column, petals, andmedial and lateral sepals were separated from each Bu. patens flower. They wereweighed and soaked individually in ethanol (more than two months). A portionof each extract was subjected to GC quantification. This was performed for fourseparate flowers (A: collected on September 12, 1995; B–D: December 9, 1996).

Feeding Stimulant Bioassays. Synthesized zingerone was tested at differentconcentrations (10 ng to 20 mg/ spot) on a TLC plate. A 1-ml solution containingthe required amount of the chemical was spotted, and it was covered with a finemist of distilled water before placing the TLC plate in a cage (43 × 43 × 43cm) containing ca. 100 mature males of B. cucurbitae or B. papayae for 15 min.If no fly was attracted to the spot, the next higher concentration was used and

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the spotting procedure repeated. The whole process is repeated until a fly wasattracted and fed on the chemical spot.

Quantification of Volatiles After Feeding on Flower or Zingerone. Sexu-ally mature male flies of B. papayae (14–16 days after eclosion), B. carambolae(25–30 days old), and B. cucurbitae (14–16 days old) were allowed to feed onBu. patens flowers for 15 min to 1 hr and then isolated in a small cage (9 cmdiam. × 15 cm long) containing fly food. The whole body of flies at 24 hr aftertreatment was soaked individually in ethanol (500 ml/ male) and kept in a freezeruntil chemical analyses. Portions of the extracts were analyzed by GC (quantifi-cation) and GC-MS (identification). At least five replicates were performed foreach treatment.

In order to examine the selective incorporation of zingerone in the body tis-sues of each species, mature males were allowed to feed ad libitum on syntheticzingerone (0.1 mg of the intact chemical placed on a glass plate in a disposablePetri dish) for 30 min and then kept in a small cage containing water. Flies at6 hr and 1 day after treatment were dissected to remove crop and rectal gland.Two crops or two rectal glands were combined and extracted with ethanol (500ml/ male). A 1-ml portion of each sample was subjected to GC quantification.Three replicates were performed for each of the organs.

RESULTS

Fruit Fly Behavior in Removal of Pollinia and Pollination

Field observations showed that the Bu. patens flower is unique amongorchids in attracting only fruit flies belonging to several species in the genusBactrocera. Male flies of B. caudata (Fabricius), B. cucurbitae, B. papayae, andB. tau were attracted and landed on the black wire-mesh enclosing a Bu. patensflower in full bloom. The floral buds, however, did not attract flies. A male fruitfly, guided by the specific flower fragrance, alighted usually on one of the lateralsepals and began feeding along the sepal. When a fly moved onto the see-sawlip, which is on a higher plane than the floral column (a characteristic of non-resupinate orchid flowers), it continued to feed as it moved slowly along the liptowards the column. Due to the shifting of the fly’s weight past the floral hinge(fulcrum), the see-saw lip would suddenly tilt towards the column, toppling thefly into the column cavity. A small ridge (callus) along the center and near thebase of the lip ensured that the fly would not fall on its side. The fly becamesqueezed between the closed lip and the column of the flower and struggledto free and/ or right itself by moving backwards. In so doing, it pushed the lipaway from the column and at the same moment touched the sticky vicidium andremoved the pollinarium, a structure consisting of pollinia, stipes, and vicidium.The pollinarium would usually stick dorsally to the fly’s thorax (Figure 1). When

ORCHID SYNOMONE FOR FRUIT FLIES 539

the fly next visits another Bu. patens flower, the protruding pollinia on the fly’sdorsum may be deposited on the stigma (at the top of the columnar cavity). Thisis probably performed by repeating the lip movements and the fly’s reaction aspreviously described for the removal of the pollinarium.

Male flies were observed to feed on the flower during the daytime. Theflowers attracted flies for three days. If the pollinia had already been removed, thecolumn cavity was normally filled with a sticky, clear fluid. When a fly came intocontact with this fluid after it was toppled into the column cavity, the fly wouldthen spend the next 10–35 min removing the sticky fluid from its compound eyesor other parts of its head and thorax. It was observed that the flower partiallyclosed in the night, as if it had began to wilt. The next morning, however, thepetals and sepals were well spread out again.

One to four male flies of several species were observed feeding at any onetime between dawn (07 : 15 hr) and dusk (19 : 00 hr) for the first three days ofblooming. No females visited the blossoms. In the wild, males of ME-sensitive(B. papayae) and RK-sensitive species (B. caudata, B. cucurbitae, and B. tau)were attracted to the flowers. In addition, a male fly with intermediate morphol-ogy between B. papayae and B. carambolae was also attracted to the flower.In the laboratory, males of B. papayae, B. carambolae, and B. cucurbitae wereattracted to and fed on the ventral surfaces of floral parts.

Identification of Male Attractants in Bu. patens Flowers

A single compound that attracted both ME- and RK-sensitive flies wasfound at a retention time (Rt) of 10.74 min in the gas chromatogram of anethanolic extract of the flowers (Figure 2). It was purified from column chro-matographic fraction 6, followed by HPLC separation (isolated at Rt c 10.20min; yield: 84 mg from two flowers). This compound was identified as zingerone[4-(4-hydroxy-3-methoxyphenyl)-2-butanone] (3) from its mass and 1H NMRspectra and by comparing the data with an authentic sample synthesized accord-ing to the method reported by Nomura (1917).

Compound 3 (Zingerone). MS: m/ z(%) 194(56), 151(16), 137(100),122(11), 119(17), 91(17), 77(10), 43(32). 1H NMR (CDCl3): d 2.13 (3H, sin-glet), 2.72 (2H, multiplet), 2.81 (2H, multiplet), 3.87 (3H, singlet), 5.46 (1H,broad singlet), 6.67 (1H, double doublet, J c 1.8, 8.0 Hz), 6.69 (1H, doublet, Jc 1.8 Hz), 6.82 (1H, doublet, J c 8.0 Hz).

Gas chromatography (Figure 2) of crude extracts of Bu. patens flowersshowed that zingerone was accompanied by a small amount of another relatedcompound (4) at Rt c 11.0, which was isolated from column chromatographicfractions 8 + 9 (6.5 mg from two flowers). It was identified as zingerol [4-(4-hydroxy-3-methoxyphenyl)-2-butanol] by comparison with a synthetic sample(prepared by reduction of 3 with lithium aluminium hydride in ether).

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Compound 4 (Zingerol). MS: m/ z(%) 196(73), 178(10), 163(18), 138(65),137(100), 131(45), 123(18), 91(24), 77(10), 45(20), 43(29).

A survey of the flower extracts also revealed the presence of methyl eugenol(1) (Figure 2, Rt c 7.52 min) in extremely low quantity (ca. 40 ng/ flower, cor-responding to a thousandth of zingerone).

Compound 1 (Methyl Eugenol). MS: m/ z(%) 178(100), 163(34), 147(26),107(21), 105(15), 103(21), 91(27), 77(15).

Contents of Zingerone in Floral Parts

Figure 3 shows results of GC quantifications of flower parts from four indi-vidual flowers (A–D). The quantity of zingerone varied greatly from flower toflower, ranging between 10 and 90 mg per whole flower. The difference in totalquantities may be due to the flower age (all collected without exposure to the fruitflies). Lateral sepals were consistently found to contain the highest quantities.However, based on concentration per fresh weight of flower parts, the sea-sawlip contained the highest concentration, e.g., contents in individual floral partsin sample D: 9.3 mg/ 10 mg lip (930 ppm); 0.2 mg/ 55 mg column (4 ppm); 2.4mg/ 47 mg petals (51 ppm); 12.1 mg/ 127 mg lateral sepals (95 ppm); and 8.7mg/ 52 mg of medial sepal (167 ppm).

FIG. 3. Contents of zingerone in each flower part of Bulbophyllum patens (from left:lip, column, petals, lateral sepals, median sepal). All samples (A–D) were collected andextracted without being exposed to fruit flies.

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Behavioral Responses of Flies to Floral Volatiles

In the laboratory, sexually mature males of B. papayae responded to a min-imum dose of zingerone (3) at 8 mg/ spot, while B. cucurbitae responded to 2mg/ spot. The males clearly detected the chemical from a distance (ca. 20 cm)and licked the chemical sources as soon as they landed near the spot. Most of themales salivated on the zingerone spot and kept feeding for more than a minute.B. carambolae males were attracted and fed on a spot (30 mg/ spot), althoughthe minimum threshold dosage was not determined. Zingerol (4) appeared to bemuch less significant in attracting B. cucurbitae. At 20 mg/ spot, it attracted malesof B. papayae at a distance of >2 m. It also attracted B. papayae females at ahigher dose (100 mg/ spot) and at closer range (within 50 cm) but did not stimu-late licking activity. Although the quantity of ME (1) in the flower was extremelylow, it may function as an attractant in addition to zingerone. B. papayae maleswere attracted to ca. 1 ng ME/ spot in the field and laboratory. However, B.carambolae males were not sensitive to the ME spot at the same dose.

Accumulation of Floral Volatiles in Rectal Glands

After feeding upon the floral tissues of Bu. patens, the male flies accumu-lated the specific fragrance in the body (Figure 4). Males of both B. carambolaeand B. papayae (ME-sensitive) stored 1–2 mg of zingerol and small quantities ofzingerone in their body. In contrast, males of B. cucurbitae (RK-sensitive) accu-mulated a large quantity of zingerone and a relatively small quantity of zingerol,almost in the same ratio as in the flower (Figure 4).

The feeding tests were also conducted with a pure sample of zingerone.

FIG. 4. Contents of zingerone (3) and zingerol (4) in body tissue extracts of three fruitfly species (Bactrocera papayae, B. carambolae, and B. cucurbitae) 24 hr after feedingon the flower of Bulbophyllum patens (average from five males).

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TABLE 1. ZINGERONE (3) AND ZINGEROL (4) IN CROP AND RECTAL GLAND OF

Bactrocera papayae, B. carambolae, AND B. cucurbitae) 6 HR AND 1 DAY AFTER

FEEDING ON ZINGERONE

Content (mg/ male)a

Crop Rectal gland

Zingerone Zingerol Zingerone Zingerol

B. papayae6 hr 8.73 ± 5.59 0.05 ± 0.08 0.01 ± 0.01 1.48 ± 0.711 day 10.37 ± 6.77 5.75 ± 2.05 0.28 ± 0.20 1.08 ± 0.58

B. carambolae6 hr 8.99 ± 3.41 0.14 ± 0.09 0.34 ± 0.19 2.07 ± 1.561 day 2.46 ± 0.66 0.30 ± 0.15 0.07 ± 0.08 1.53 ± 0.72

B. cucurbitae6 hr 10.91 ± 6.58 0.12 ± 0.17 3.66 ± 0.91 0.36 ± 0.021 day 9.23 ± 1.38 0.53 ± 0.39 10.34 ± 2.09 0.86 ± 0.09

aAmounts are average ± SD, N c 3.

Table 1 shows the quantities of zingerone and zingerol present in the crop andrectal glands after feeding on zingerone (dissected 6 hr and 1 day after ingestion).In both B. carambolae and B. papayae, zingerone was found in the crop and aportion of zingerol was selectively stored in the rectal gland. A large proportionof zingerol was present in the crop of B. papayae males one day after treatment,suggesting the crop as the site of conversion via chemical reduction. In contrast,B. cucurbitae males stored a large quantity of zingerone with a small amount ofzingerol in the rectal gland (zingerone–zingerol 10 : 1) (Table 1).

DISCUSSION

Zingerone is the first example of an attractant that exerts activity both onthe ME- and RK-sensitive Bactrocera fruit flies. Zingerone (3) possesses somestructural resemblance to both ME (1) and RK (2) (Figure 2). This duality maybe of ecological importance for the plants to secure fruit fly pollinators as soonas possible, yet select pollinators within a specific insect group in a forest thatcontains a high diversity of organisms, especially during the orchid floweringseason (February–April). This clearly contrasts with the case of orchid–bee asso-ciations, where each orchid species is associated primarily with a single or a fewbee species (Dressler, 1982; Whitten et al., 1989; Borg-Karlson, 1990). Eventhough the zingerol and zingerone from male rectal glands stimulate female sex-ual activity (Tan, 1998), females have not been observed to be attracted to the

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flowers, probably due to the low quantity released and the short-range attractingnature of the compounds to females.

Males of these Bactrocera species emit rectal volatiles as a pheromonesmoke at dusk, the time when mating takes place (Ohinata et al., 1982; Kuba andSokei, 1988). Males fed on ME or other attractant phenylpropanoids selectivelystore the intact chemicals or their metabolites in the rectal glands and releasethem during the courtship period (Nishida et al., 1988, 1990, 1993; Nishida andFukami, 1990). Males fed on the phenylpropanoids are more successful in matingthan unfed males in B. dorsalis (Hendel), B. philippinensis Drew & Hancock,B. papayae, and B. cucurbitae (Shelly and Dewire, 1994; Shelly and Villalo-bos, 1995; Shelly et al., 1996, Nishida et al., 1997; Tan and Nishida, 1998).The rectal metabolites (trans-coniferyl alcohol and 2-allyl-4,5-dimethoxyphe-nol) play a role as sex pheromones to attract and arrest conspecific females ofB. dorsalis (Hendel) and B. papayae (Tan and Nishida, 1996, 1998; Nishidaet al., 1997; Hee and Tan, 1998; Tan, 1998). Likewise, zingerone and zingerolsequestered in the rectal glands by the flies appear also to act as sex pheromonalcomponents. In our preliminary observations, synthetic zingerol (20 mg) attractedfemales of B. papayae at close range in a cage and in a wind tunnel. An attractedfemale extruded its ovipositor (a mating acceptance posture) toward the chemicalsource. Therefore, the rectal zingerol potentially functions as the sex pheromoneat least in B. papayae, whenever a naturally releasing chemical source in suffi-cient quantities is available in nature. Although the pheromonal role of zingerolin B. carambolae has not been determined, an additional endogenous volatile (6-oxo-1-nonanol) present in the rectal gland (Perkins et al., 1990) may be involvedfor discrimination of species (Wee and Tan, unpublished data). In B. cucurbitae,zingerone was found to act as a male sex pheromone component, attracting sex-ually mature females at close range (Tan, 1998).

In B. papayae feeding on ME, the rectal substances (trans-coniferyl alcoholand 2-allyl-4,5-dimethoxyphenol) were suggested to play a role as an aggrega-tion pheromone, attracting males to form a lek in the initial stage of courtship(Hee and Tan, 1998). The same activities were observed in the zingerone-fed males of B. cucurbitae in a wind tunnel, where the males induced sig-nificantly more conspecific males and females to fly upwind than zingerone-deprived males. In a wind tunnel, zingerone-fed males attracted 20–50% females,with a peak at 17 : 30–18 : 00 hr, compared with <10% for deprived malesbetween 17 : 00 and 19 : 00 hr. They attracted conspecific males earlier, peak-ing at 17 : 15–17 : 45 hr, compared with deprived males having peak attractionafter 18 : 00 hr (Khoo and Tan, unpublished).

ME metabolites (trans-coniferyl alcohol and 2-allyl-4,5-dimethoxyphenol)sequestered by B. papayae also play a significant role as defense substancesagainst bird predators and geckos (Nishida et al., 1988; Jakubas et al., 1992; Tan,1994; Tan and Nishida, 1998). Zingerone is a natural pungent-tasting, irritant

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chemical found in ginger rhizomes [Zingiber officinale Rosc. (Zingiberaceae)](Liu and Simon, 1996). It evokes many of the same physiological responsesin man and other mammals as its structurally related compounds capsaicin andpiperine, found in chilli (Capsicum annum L.) and black pepper (Piper nigrumL.), respectively (Liu and Simon, 1996). Therefore, zingerone, besides actingas a pheromone, may also act as an irritant or deterrent to potential vertebratepredators. The roles of sequestered zingerone and zingerol by Bactrocera malesin the defense mechanism certainly warrant further studies.

Acquisition of the phenylpropanoids may have evolved initially in the con-text of sexual selection, particularly female preference for males scented withchemicals derived from flowers (Nishida et al., 1997). In terms of coevolutionbetween fruit flies and plants, zingerone in Bu. patens may mark the point atwhich divergence of natural fruit fly attractants occurs in the plant kingdom. Onthe contrary, it may be a result of manipulation of the phenylpropanoid moleculeby the plant to “convergently” attract two groups of fruit fly species (ME- andRK-sensitive) in the complex tropical ecosystem. Raspberry ketone occurs in thefloral fragrance of the raspberry jam orchid, Dendrobium superbum (D. anos-mum) Lindl. (Nishida et al., 1993), and the flower attracts only RK-sensitivespecies such as B. albistragata (de Meijere), B. caudata, B. cucurbitae, and B.tau. In this case, however, the male flies feed on the petals and sepals and noflies enter the lip cavity. The Bu. patens–fruit fly association provides a uniquecase of coevolution in which both organisms have reproductive benefits arisingfrom the close relationship.

The Bactrocera genus of dacine fruit flies includes many serious pestspecies that infest tropical and subtropical fruit species. ME and cue-lure (acetyl-RK) have been successfully employed in control and eradication programs ofthese destructive pests (Chambers, 1977; Koyama et al., 1984). Zingerone mayalso be effectively introduced in the management of these fruit flies. Unfortu-nately, the use of these attractants could threaten the existence of Bu. patensin the natural habitat if applied to a vast area and the fruit fly pollinators wereeliminated.

Acknowledgments—We thank A. Lamb, Agricultural Research Station, Tenom, Sabah, andS. T. Tan, USM, for assistance and orchid plants, and our universities for providing research facilities.This work was supported by the Malaysian Government IRPA grant MPKSN/ IRPA 01-02-05-6004and Grant-in-Aid for Scientific Research (No. 10460049) from the Ministry of Education, Scienceand Culture of Japan.

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