Journal of Chemical Ecology, Vol. 30, No. 11, November 2004 (©C 2004)

MALE SEX PHEROMONAL COMPONENTS DERIVED FROMMETHYL EUGENOL IN THE HEMOLYMPH OF THE FRUIT

FLY Bactrocera papayae

ALVIN KAH-WEI HEE1,∗ and KENG-HONG TAN2

1School of Biological Sciences, Universiti Sains Malaysia11800 Minden, Penang, Malaysia

2Tan Hak Heng Co., 20, Jalan Tan Jit Seng11200 Tanjong Bunga Penang, Malaysia

(Received May 24, 2004; accepted August 6, 2004)

Abstract—Pharmacophagy of methyl eugenol (ME)—a highly potent maleattractant, by Bactrocera papayae results in the hydroxylation of ME to sexpheromonal components, 2-ally-4,5-dimethoxyphenol (DMP) and (E)-coniferylalcohol (CF). These compounds, which are also male attractants, are then se-questered and stored in the rectal gland prior to their release during courtshipat dusk. Chemical analyses of the digestive tract (excluding the crop and rectalgland) showed the absence of the sex pheromonal components and their precur-sor, ME. However, B. papayae males were attracted to and fed on the ME-fedmale hemolymph extracts but not on hemolymph extracts of ME-deprived males.After thin layer chromatography in a hexane:ethyl acetate solvent system, flieswere attracted to and fed on the original point on the TLC plate where thehemolymph extract had been spotted, suggesting that the pheromone compo-nents were bound in polar complexes. Chemical analyses of the ME-fed malehemolymph and crop extracts revealed the presence of the sex pheromonalcomponents. The presence of the ME-derived pheromonal components and theabsence of ME in the hemolymph suggest that the hemolymph is involved inthe transportation of sex pheromonal components from the crop to the rectalgland.

Key Words—Bactrocera papayae pharmacophagy, methyl eugenol, 2-allyl-4,5-dimethoxyphenol, (E)-coniferyl alcohol, sex pheromone, sequestration,hemolymph, rectal gland, crop.

∗ To whom correspondence should be addressed. Current address: National Center for Advanced Bio-Protection Technologies c/o HortResearch, Canterbury Agriculture and Science Center, Gerald StreetPO Box 51, Lincoln 8152, Canterbury, New Zealand. E-mail: ahee@hortresearch.co.nz

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0098-0331/04/1100-2127/0 C© 2004 Springer Science+Business Media, Inc.

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INTRODUCTION

The fruit fly Bactrocera papayae (Drew & Hancock) (Diptera: Tephritidae), asibling species of the Oriental fruit fly, B. dorsalis (Hendel) is one of the mostdestructive fruit fly pests of commercial fruits in the southeast Asian region,particularly Malaysia, Indonesia, and Thailand. Because of the destructive natureof these two congeners, there has been considerable study of their taxonomicstatus, as both species readily interbreed to produce viable offspring, and both haveidentical sex pheromonal components derived from methyl eugenol (ME) (Tan,1993, 2000; Tan and Nishida, 1996, 1998). Recent data suggests that B. papayaeand B. dorsalis may not be distinct genetic species but possibly strains of the samespecies (Naeole and Haymer, 2003).

Males of B. papayae are strongly attracted to and compulsively feed onmethyl eugenol (ME). Consumption of ME by males of B. papayae has beenfound to enhance their mating competitiveness (Tan and Nishida, 1996, 1998; Heeand Tan, 1998). A similar phenomenon was also shown to occur with males ofB. dorsalis (Shelly and Dewire, 1994; Shelly, 1995). In B. papayae, male fliesconvert the ingested ME to other phenylpropanoids, 2-allyl-4,5-dimethoxyphenol(DMP) and (E)-coniferyl alcohol (CF) (Nishida et al., 1988; Tan and Nishida,1996). These metabolites are sequestered in the male rectal gland and subsequentlyreleased during courtship at dusk. ME itself has never been detected in the rectalgland of ME-fed males (Nishida et al., 1988; Tan and Nishida, 1996). Behavioralstudies have further demonstrated that both DMP and CF function as male sex andaggregation pheromones for B. papayae (Hee and Tan, 1998), henceforth referredto as sex pheromonal components.

Whereas the ecological relationship between the attractant, ME and the fruitfly, B. papayae, has been shown to involve the production of bioactive derivativesfrom ME as pheromonal components, little is known about the physiological fateof these bioactive compounds in relation to pheromone communication, espe-cially in the production and transportation of these compounds to the rectal glandwhere release occurs. Here, we report the presence of the male sex pheromonalcomponents derived from ME in the hemolymph and crop of male B. papayae.

METHODS AND MATERIALS

Insects. A colony of B. papayae (12 generations per year) was originallycollected from infested starfruits, Averrhoa carambola L. in 1995, and culturedusing an artificial diet as described by Hee and Tan (1998). Adult flies were fed adiet containing yeast, sugar, protein, and water. Males and females were segregated1–3 d after emergence (DAE). Flies were maintained under ambient conditions inan insectary with a 12L:12D cycle and 83–90% RH at 25–29◦C. Sexually maturevirgin males (14–20 DAE) that responded maximally to ME (Tan et al., 1987)

MALE SEX PHEROMONAL COMPONENTS DERIVED FROM METHYL EUGENOL 2129

were used for experimentation. All experiments on males were performed in themorning during the peak period (0830–1100 hr) of response to ME (Tan, 1985).

Chemicals. ME (purity >98%) was purchased from Merck-Schuchardt,Germany. Authentic standards of 2-allyl-4,5-dimethoxyphenol (DMP) and(E)-coniferyl alcohol (CF) (96% trans) were synthesized and provided byR. Nishida, Kyoto University, Japan. An emulsion of ME at a concentration of26.3 mg per 100 µl of 1% Tween 80 (polyoxyethylene sorbitan monooleate)solution was prepared for feeding male flies.

Consumption of Methyl Eugenol. A male fly was placed with ventral side upand wings held firmly with plasticine. It was directly fed with 0.5 µl (containing105 µg) of ME using a capillary micropipette. To avoid direct contact with otherbodily parts, a small piece of plastic netting was placed across the legs.

Gas Chromatography-Mass Spectrometry (GC-MS). Methods employed inidentification of sex pheromonal components were based on those used by Nishidaet al. (1988). GC-MS analyses were performed on an HP 5989B mass spectrometer(electron impact, at 70 eV) connected to a non-polar GC column (30 m × 0.25 mmHP-5MS, 0.33-µm film thickness) programmed from 80◦C (1-min hold) to 240◦Cat 10◦C/min. Compounds were identified by comparison of retention times andmass spectra with those of authentic standards.

GC quantifications of sex pheromonal components were done on a ShimadzuGC-14A gas chromatograph using an HP Ultra-1 capillary column (25 m ×0.2 mm, 0.33-µm film thickness), with the same temperature program as above,calibrating with authentic standards of known concentrations, using a C-R6Aintegrator (Shimadzu).

Extraction of Digestive Tract

Gut Tissues. Dissection to remove the digestive tract was performed as de-scribed by Hee and Tan (2000). The digestive tract (excluding the crop and rectalgland) from 3 ME-fed males was extracted in Clarke insect saline 30 min afterinitial feeding on ME; sex pheromonal components were already detectable inthe rectal gland within this time (unpublished data). For chemical analyses, 10 µlof ethanol were added to the dissected tissues as solvent, whereas for detectionof bioactive samples, 10 µl of Clarke insect saline solution were used instead.One-microliter injection samples were used. A similar procedure was repeatedusing ME-deprived males as control.

Crop Tissues. The crop of a sexually mature virgin ME-fed male was dissected5 min after initial feeding with ME and subjected to several rinses of saline, water,and ethanol to remove traces of contamination. The time of crop dissection waschosen because in vitro, crop extracts of B. papayae male are able to rapidlyconvert ME to sex pheromonal components within 2–5 min (Lim et al., 1998).This procedure was repeated for another 9 crop organs. A total of 10 crops were

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homogenized with 30 µl of saturated NaCl solution and 15 µl of redistilled ethanol.The homogenate was then subjected to thorough mixing and ultrasonication.Then, 20 µl of benzene:ethyl acetate (3:1, v/v) were added and the mixturewas thoroughly mixed and centrifuged at 1000 g for 5 min. The aqueous phasewas re-extracted with 20 µl of benzene:ethyl acetate solution and the combinedorganic solution was concentrated under a gentle nitrogen flow to approximately5 µl. One and two microliter portions of the sample were used for GC and GC-MS injections, respectively. As controls, crop samples were extracted in a similarprocedure from ME-deprived males.

Hemolymph Extraction. Hemolymph was obtained from sexually maturevirgin ME-fed males 30 min after initial feeding on ME through a small longitu-dinal slit made along the center of the meso- and metathoracic segments, usinga disposable glass capillary micropipette with a drawn-out tip. On average, ca.0.5 µl of hemolymph were collected from each male. Approximately 15–20 µlof hemolymph were accumulated in a 0.2-ml microcentrifuge tube (on ice) con-taining trace amounts of phenylthiocarbamide (1-phenyl-2-thiourea) to inhibittyrosinase activity. The combined hemolymph was then centrifuged at 12,000 gfor 15 min at 4◦C. As control in the experiment, the procedure was repeated usingME-deprived males. A new micropipette was used for each set of hemolymphcollections to prevent cross contamination. For GC and GC-MS analyses, 2 µl ofthe hemolymph were added to 10 µl of ethanol and 1-µl aliquots of the mixturewere used for GC-MS and GC injections.

Attraction of Male B. papayae to Extracts of Digestive Tract and Hemolymphof ME-Fed Conspecific Males. To ascertain whether the digestive tract (excludingthe crop and rectal gland) and/or hemolymph contained the sex pheromonal com-ponents, a simple bioassay was devised. The bioassay worked on the principle thatsexually mature males of B. papayae are attracted to the sex pheromonal com-ponents at quantities often undetectable by GC (Nishida et al., 1988; Tan, 2000).Therefore, attraction of the males to and feeding on the digestive tract and/orhemolymph extracts would indicate the possible presence of the pheromonalcomponents before subjecting the positive extract for further chemical analyses.Male flies used in this method had not been exposed to DMP or CF, because ex-posure may induce habituation to the pheromonal compounds (Khoo et al., 2000).Two microliters each of the ME-fed male digestive tract and hemolymph extractswere separately dispensed as two spots on a Whatman No. 1 filter paper (diam11.0 cm) using Drummond micropipettes (32 mm). The filter paper was placedin a cage (43 × 43 × 43 cm) containing 100 mature males of B. papayae for15 min. The attraction and behavior of the flies were observed. Extracts attrac-tive to males were then subjected to thin layer chromatographic analyses. In thecontrol experiment, the procedure was repeated using extracts from ME-deprivedmales. Three replicates were conducted using different batches of males to avoidhabituation to the volatiles.

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Thin Layer Chromatography (TLC) and Assay of Bioactive Spots. Portionsof the bioactive extracts (10 µl) previously identified as attractive to flies weresubjected to TLC on a precoated plate (HPTLC, silica gel 60 F254, nano TLC,Merck) and developed with hexane:ethyl acetate (2:1, v/v) prior to visualizationunder UV at 254 nm. As controls, authentic DMP and CF (each 1 µl of 10 mg/mlsolution) were spotted separately on different lanes. All samples were allowedto dry completely before being developed. Spots indicating DMP and CF weremarked with pencil and covered with a small piece of 3M ScotchTM tape to preventattraction of males in the subsequent detection of active spots. The developedplate then was slightly moistened by misting with deionized water to enhance theevaporation rate of volatiles (Hee and Tan, 1998). The plate was introduced intoa cage (43 cm × 43 cm × 43 cm) containing 100 sexually mature flies. Theirattraction and behavior were observed for 15 min to determine the active zones.Three replicates were performed each time using different batches of flies.

Separation of Hemolymph Extracts in the Original Spot on the TLC Plate.Following development on the TLC plate containing ME-fed male hemolymphwith hexane:ethyl acetate (2:1, v/v), the original spot on the TLC plate (foundpreviously to be attractive to conspecific males) was allowed to dry completely.Then, the original spot was scraped into a glass tube (Tan, 1972). One ml of ethanolwas added to the silica gel in the tube and the mixture was thoroughly stirred, thencentrifuged at 3000 g for 10 min. The resulting supernatant was transferred to anew tube, and 1 ml of ethanol was added to the pellet. This procedure was repeatedtwice. The accumulated supernatant was then concentrated further under partialpressure (Buchi rotary evaporator) to near dryness. The concentrated samplewas resolubilized in 30 µl of ethanol for GC-MS and GC analyses, injecting1-µl aliquots. As controls, ME-deprived male hemolymph was subjected to theprocedure. The experiment was performed in triplicate.

RESULTS

Male Attraction to Extracts of Digestive Tract and Hemolymph of ME-FedMales. Extracts of the digestive tract (excluding the crop and rectal gland) ofME-fed male B. papayae flies when presented to sexually mature males did notelicit any attraction or feeding activity. Subsequently, ME and sex pheromonalcomponents were not detected in analyses of the digestive tract extracts. However,male flies were attracted to the spot on the TLC plate containing hemolymph fromME-fed males, exhibiting rapid short zig-zag flight before landing and feeding onthe hemolymph spot. In contrast, there was no attraction to hemolymph extract ofME-deprived males (Figure 1).

Thin Layer Chromatography (TLC) and Biodetection. The developed TLCplate containing extracts of hemolymph from ME-fed males did not show the pres-ence of pheromonal components when viewed under UV illumination at 254 nm

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FIG. 1. Attraction of sexually mature virgin males of Bactrocera papayae to hemolymphextracts. (A) Males feeding on spot containing hemolymph extracts of methyl eugenol-fed(ME-FED) males. (B) No attraction of males to spot marked “X” containing hemolymphextracts of methyl eugenol-deprived (ME-DEP) males.

(Figure 2A). However, when the plate was exposed to mature male flies, attractionto and feeding on the original spot were observed as compared with no attrac-tion to the adjacent spot containing hemolymph extract from ME-deprived males(Figure 2B). To discount the possibility that the original spot was overwhelmingthe attraction of males to other areas on the TLC plate, the original spot was thencovered with a piece of scotch tape. Following further exposure of the plate tomale flies, little (1–2 males) or no attraction of males to the spots with Rf valuescorresponding to DMP and CF on the plate was observed.

Gas Chromatographic Analyses. GC analyses confirmed the presence of thesex pheromonal components in the hemolymph extract of ME-fed males (Figure 3).Pheromonal components were identified from matches in retention time and massspectra with authentic standards of DMP and CF. ME was not found.

The sex pheromonal components and ME were also detected in the cropextract of ME-fed males (Figure 3B). In addition, after TLC, DMP and CF werealso found in the ME-fed male hemolymph extract, both in the original spot andin the spots having similar Rf value to DMP (Rf = 0.45) and CF (Rf = 0.26),respectively (Table 1).

DISCUSSION

ME is found in at least 200 species of plants from 32 families, and hasbeen shown to play a central role in the chemical ecology of ME-sensitive fruit

MALE SEX PHEROMONAL COMPONENTS DERIVED FROM METHYL EUGENOL 2133

FIG. 2. (A) Thin layer chromatogram of hemolymph spots from methyl eugenol-fed (ME-FED) and -deprived (ME-DEP) males. Separated spots of authentic standards of CF andDMP are shown on the right. Note that CF and DMP were not detected in hemolymphextracts of ME-fed and -deprived males. (B) Attracted males feeding on original spotcontaining the methyl eugenol-fed male hemolymph (left) and no attraction to spot marked“X” containing hemolymph of methyl eugenol-deprived male (right) following thin layerchromatography.

fly species (Tan, 1993, 2000; Tan and Nishida, 1998). Consumption of ME andits conversion to DMP and CF in ME-fed male flies has been shown to con-fer protection against vertebrate predators (Nishida and Fukami, 1990; Tan andNishida, 1998; Wee and Tan, 2001). Further, ME and DMP have also been de-tected from Bulbophyllum orchids such as Bu. cheiri (Tan et al., 2002; Nishidaet al., 2004). In Bu. vinaceum, both DMP and CF were detected along with ME

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FIG. 3. Gas chromatograms of sex pheromone components present in (A) hemolymph and(B) crop extract (in trace amounts) of methyl eugenol-fed Bactrocera papayae males. (1)2-allyl-4,5-dimethoxyphenol, (2) (E)-coniferyl alcohol, and (3) methyl eugenol.

as major components in floral fragrance (Tan, K. H. and Nishida, R., unpublisheddata). The interrelationship and coevolution of this genus of wild orchids andME-sensitive Bactrocera species are currently being examined (Tan and Nishida,2000; Tan et al., 2002).

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TABLE 1. SEX PHEROMONE COMPONENTSa RECOVERED FROM

HEMOLYMPHb OF METHYL EUGENOL-FED Bactrocerapapayae MALES

Content (ng)∗

DMP CF

Original spot 78 ± 26 87 ± 26Spot corresponding to DMP (Rf = 0.45) 17 ± 14 —Spot corresponding to CF (Rf = 0.26) — 13 ± 8

aAfter thin layer chromatography (HPTLC, silica gel 60 F254, nano TLC,Merck) using a solvent system of hexane:ethyl acetate (2:1, v/v).

b10 µl of hemolymph was dispensed on the original spot.∗Means ± SE, N = 3.

This investigation marks the first time that ME metabolites, which func-tion as sex pheromonal components, were detected in both the hemolymph andcrop organ of B. papayae males following consumption of ME. Further stud-ies (unpublished data) have also suggested that hemolymph transports DMPand CF, as indicated by the presence of possible peptide carriers in hemolymphfractions containing pheromone components. These results suggest that the sexpheromonal components are biosynthesized from a common precursor, ME, inthe crop and transported probably by the hemolymph carrier(s) to be sequesteredby the male rectal gland. Further light microscope studies have demonstratedthe accumulation of oil droplets and autofluorescent compounds in the rectalpapillae and sacs of ME-fed males (Khoo and Tan, personal communication).If these oils contain the pheromone components, then the data suggest that thepheromones are transported by hemolymph carriers to the rectal gland via the rectalpapillae.

In contrast, endogenously produced terpenoid sex pheromonal componentsin the hemolymph, crop, and oral secretions of the male Caribbean fruit fly,Anastrepha suspensa (Loew) were isolated and identified (Teal et al., 1999; Luand Teal, 2001). The principal storage/release sites of these terpenoids werethe salivary glands, hindgut, anal pouch, and the pleural abdominal glands,which are exposed during courtship displays (Nation, 1990). In addition, thehemolymph of A. suspensa has also been suggested to play a role in the transportof these terpenoid sex pheromonal components during sexual signalling (Teal et al.,1999).

Bioassays using B. papayae males to detect the presence of ME-derivedsex pheromonal components in extracts of the digestive tract (excluding the cropand rectal gland) and hemolymph of ME-fed males were shown to be effective,sensitive, reliable, and economical. This technique enables preliminary screeningfor the presence of pheromonal components prior to chemical analyses, that are

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often tedious to perform and sometimes less sensitive. Although females are knownto be attracted to the sex pheromonal components (Nishida et al., 1988; Tan andNishida, 1996, 1998; Khoo et al., 2000), female attraction has been difficult toobserve because when a female is attracted, the fly would be moving, rapidlyat times, near the attractant spot. Furthermore, at low doses, females were notobserved to extrude their ovipositors (Tan and Nishida, 1996, 1998). Thus, it isdifficult to pinpoint the exact spot that female flies were attracted to. In contrast,the use of male flies facilitates the detection of bioactive hemolymph samplesbecause males feed on the spot containing the pheromonal components and leavea distinct salivary mark.

In addition, males have a wider temporal response window than females. Thisis attributed to the fact that the period during which males are most sensitive to thepheromonal components occurs between 0800 and 1100 hr (Tan, 1985; Nishidaet al., 1988; Tan and Nishida, 1996, 1998; Khoo et al., 2000; Wee et al., 2002),whereas that of females is only half an hour, between 1845 and 1915 hr (Hee andTan, 1998; Khoo et al., 2000).

The behavioral pattern of B. papayae males attracted to the extracts ofhemolymph of ME-fed males is similar to that of ME, i.e., a sequence of zig-zaganemotaxis toward the source of ME and pulsation of the mouthparts followed bycompulsive feeding on ME (Metcalf et al., 1975). DMP and CF are also highlyattractive to male flies and elicit a searching behavior similar to that of malesto ME (Nishida et al., 1988; Tan and Nishida, 1998; Tan, 2000). Recent probitanalysis of B. papayae male quantum response to ME showed ED50 (effectivemedian dose required to elicit response in 50% of the fruit fly population tested)of 318 ng (Wee et al., 2002). If ED50 of DMP and CF are assumed to be withinME range, the low quantity of DMP and CF in the corresponding spots on TLC(having similar Rf values of authentic samples) would attract none or 1–2 males,as evidenced from these results.

Results from the biodetection of bioactive spots following TLC suggest thatthe solvent system of hexane:ethyl acetate (2:1; v/v) was not able to effectivelyseparate DMP and CF from hemolymph of ME-fed males. In contrast, spot ap-plications of authentic DMP and CF on the TLC plate and the subsequent chro-matographic development resulted in these samples being separated easily, andthe developed spots were detected under ultraviolet illumination. To discount thepossibility that water soluble materials from the hemolymph were interfering withthe free movement of solvent through the spot, the samples were allowed to drycompletely prior to solvent development. Therefore, the larger amounts of thepheromonal components in the original spot suggest the presence of a higher de-gree of bound pheromonal components than that of free or unbound componentsin the hemolymph. As such, the solvent partitioning effect of TLC was not able todissociate the bound pheromonal components. This suggests the occurrence of asex pheromone binding complex in the hemolymph of ME-fed males.

MALE SEX PHEROMONAL COMPONENTS DERIVED FROM METHYL EUGENOL 2137

Detection of sex pheromonal components in the crop extracts of ME-fed maleflies also suggested that it is the site of ME hydroxylation to the ME metabolites,DMP and CF. In vitro studies on crop extracts of ME-fed male B. papayae haveshown the presence of an enzyme with mixed-function monooxygenase activitybased on the cytochrome P-450 system (Lim et al., 1998). In addition, the absenceof ME in the hemolymph and digestive tract (excluding the crop) suggests thatME is completely converted into DMP and CF in the crop, or the crop membraneis impermeable to ME but ME-derived pheromonal components are probablyactively transported across the crop membrane into the hemolymph.

Acknowledgments—We thank R. Nishida, Kyoto University, for the supply of chemicals andassistance in GC-MS analyses, and Y. C. Toong and S. T. Tan (USM) for their interest and discussion.The Malaysian Government IRPA MPKSN/IRPA grant 01-02-05-9007 with working facilities providedby USM supported this work.

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