Proc. Natl Acad. Sci. USAVol. 80, pp. 3143-3147, June 1983Applied Biology

Olfactory receptors in the melon fly Dacus cucurbitae and theoriental fruit fly Dacus dorsalis

(kairomone/secondary plant substance/coevolution)

ROBERT L. METCALF*, WALLACE C. MITCHELLt, AND ESTHER R. METCALF**Department of Entomology, University of Illinois, Urbana-Champaign, Illinois 61801; and tDepartment of Entomology, University of Hawaii at Manoa,Honolulu, Hawaii 96822

Contributed by Robert L. Metcalf, March 21, 1983

ABSTRACT Male melon flies (Dacus cucurbitae) from a col-ony in Hawaii were evaluated for limit of response to the olfactorystimulant raspberry ketone and to more than 40 related mole-cules. The results were compared with the limits of response oforiental fruit flies (Dacu dorsalia) under identical conditions. Thenature of the response of the two species to attractive compoundsappeared to be identical with regard to orientation, searching,pulsating mouthparts, and compulsive feeding. However, therewas very little overlap in the response of the two species to phenyl-propanoid-type compounds. D. cucurbitae responded most stronglyto p-hydroxyphenylpropanoids while D. dorsali responded moststrongly to 3,4-dimethoxyphenylpropanoids. The results are dis-cussed in terms of antennal receptor site geometry and with re-gard to the coevolution of two major groups of Dacini with plantkairomones.

The melon fly Dacus cucurbitae Coquillette (Diptera, familyTephritidae), a native of Southeast Asia, is widespread in China,Japan, the Ryukyus, the Philippines, Thailand, New Guinea,and northern Australia and is also found in Hawaii, Mauritius,and Kenya and Tanzania (1). The sexually mature male melonfly is strongly attracted to 4-(p-methoxyphenyl)-2-butanone("anisyl acetone") (2) and to 4-(p-acetoxyphenyl)-2-butanone (cue-lure), developed as a more efficient lure attracting both teneraland sexually mature male flies (3, 4). Cue-lure is intrinsicallyless attractive than its hydrolysis product 4-(p-hydroxyphenyl)-2-butanone (Willison's lure or raspberry ketone, also named p-hydroxyphenyl-3-butanone) (5, 6). Raspberry ketone is a phen-ylpropanoid natural product found in the raspberry (7) and asa glycoside in Rhizoma rhei (8).

D. cucurbitae is a representative of the tribe Dacini com-prised of more than 1,000 tropical and subtropical species whoselarvae develop in a great variety of fruits (9). Drew and co-workers (5, 9, 10) surveyed the responses of 79 species of maleDacini in the South Pacific region and 55 species in Australia.Their data together with other references suggest that approx-imately 92 species of Dacini respond to cue-lure or Willison'slure (raspberry ketone) and 40 species respond to methyl eu-genol. No species has been shown to respond to both types oflure and all species within each complex of closely related spe-cies responded to the same lure, indicating that the olfactoryresponse has profound evolutionary implications.

Previously, we (11, 12) suggested that ancestral Dacini co-evolved with plants containing p-hydroxycinnamic acid (p-cou-maric acid), a widely distributed secondary plant compound thatis a precursor in the biosynthesis of both methyl eugenol andraspberry ketone (13-15). The two discrete groups of Daciniresponding to either methyl eugenol or raspberry ketone have

developed specific antennal receptors complementary in struc-ture to one or the other of these two phenylpropanoid kairo-mones yet both groups must have developed from a commonancestor. In the present study we compared the qualitative andquantitative responses of male fruit flies of D. cucurbitae(subgenus Zeugodacus), responsive to raspberry ketone, and ofmale D. dorsalis (subgenus Bactrocera), responsive to methyleugenol, to more than 40 related chemicals. The informationobtained is essential to characterize the antennal receptor sitesof the two species and useful in understanding their chemicalecology and their coevolutionary development with host plants.

MATERIALS AND METHODSThe odorants investigated (Table 1) were synthesized and pu-rified by recrystallization or vacuum distillation and their pu-rities and identities were confirmed by TLC, IR spectrometry,and physical properties, or if not further identified were Ald-rich "white label" quality. The basic odorant used to charac-terize the response of D. cucurbitae was 4-(p-hydroxyphenyl)-2-butanone (raspberry ketone, II; mp 82-830C) and that for D.dorsalis was 3,4-dimethoxyallylbenzene [methyl eugenol, I; bp120-122/10 mm (16)]. The 4-(p-acetoxyphenyl)-2-butanone (cue-lure, IV) was supplied by the U. S. Department of Agriculture.The benzyl acetates VIII, IX, X, XI, and XI were preparedfrom the corresponding alcohols by refluxing with excess aceticanhydride containing a catalytic amount of pyridine. This pro-cedure was effective in preparing p-acetoxybenzyl acetate (XII)from p-hydroxybenzyl alcohol (17) but p-hydroxybenzyl acetate(VII) (17, 18) rapidly polymerized and it was prepared by re-fluxing p-hydroxybenzyl alcohol with isopropenyl acetate con-taining a catalytic amount of sulfuric acid. The product was pu-rified by column chromatography on silicic acid.The carboxylic acid methyl esters XX, XXII, XXIV, XXVI,

XXVIII, XXX, XXXII, XXXIV, XXXVmI, XL, XLII, and XLIVwere prepared by refluxing the corresponding acids in excessabsolute methanol containing a catalytic amount of sulfuric acid.The phenyl propionates (XVII and XVIII) were prepared by

treating propionic anhydride with the potassium and sodiumsalts of the appropriate phenols. The alkyl ethers of 4-(p-hy-droxyphenyl)-2-butanone, anisyl acetone (L and XLV andXLVI) were prepared by treating the sodium salt with the ap-propriate alkylating agent.The limits of response (LR) of reared D. cucurbitae and D.

dorsalis 12-20 days old were measured at 20-220C by applyingthe test compounds from microliter pipettes to 9-cm Whatmanno. 1 filter paper discs with aluminum foil backing. Solutionswere prepared in reagent-grade acetone and diluted (wt/vol)on a logarithmic scale until the threshold concentration (LR)

Abbreviation: LR, limit(s) of response.

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Table 1. Responses of male D. cucurbitae and D. dorsalis to kairomone analoguesLR, .g

Analogue

3,4-(CH30)2C6H3CH2CH=CH24-HOC6H4CH2CH2C(O)CH34-CH30C6H4CH2CH2C(O)CH34-CH3C(O)OC6H4CH2CH2C(O)CH3C6H5CH2CH2C(O)CH34-HOC6H4CH2CH2CH2CH34-HOC6H4CH20C(O)CH34-CH30C6H4CH20C(O)CHg3-CH30C6H4CH20C(O)CH32-CH30C6H4CH20C(O)CH33,4-(CH30)2C6H3CH20C(O)CH34-CH3C(O)OC6H4CH20C(O)CH3C6H5CH20C(O)CH34-CH30C6H4C(O)CH34-CH30C6H4CH2C(O)CH33,4-(CH30)2C6H3CH2C(O)CH34-HOC6H40C(O)CH2CH34-CH30C6H40C(O)CH2CH34-CH30C6H4C(O)OH4-CH30C6H4C(O)OCH34-HOC6H4CH2C(O)OH4-HOC6H4CH2C(O)OCH34-CH30C6H4CH2C(O)OH4-CH30C6H4CH2C(O)OCH33,4-(CH30)2C6H3CH2C(O)OH3,4-(CH30)2C6H3CH2C(O)OCH34-HOC6H4CH2CH2C(O)OH4-HOC6H4CH2CH2C(O)OCH34-CH30C6H4CH2CH2C(O)OH4-CH30C6H4CH2CH2C(O)OCH33,4-(CH30)2C6H3CH2CH2C(O)OH3,4-(CH30)2C6H3CH2CH2C(O)OCH34-CH30C6H4CH2CH2CH2C(O)OH4-CH30C6H4CH2CH2CH2C(O)OCH34-HOC6H4CH2CH(NH2)C(O)OH (dl)4-HOC6H4CH2CH(NH2)C(°)OCH3 (L)

4-HOC6H4CH=CHC(O)OH4-HOC6H4CH=CHC(O)OCH34-CH30C6H4CH=CHC(O)OH4-CH30C6H4CH=CHC(O)OCH33,4-(CH30)2C6H3CH=CHC(O)OH3,4-(CH30)2C6H3CH=CCHC(O)OCH33-CH30-4-HOC6H3CH=CCHC(O)OH3-CH304HOC6H3CH=CHC(O)00H34-C2H50C6H4CH2CH2C(O)CH34-C3H70M6H4CH2CH2C(O)CH3

mp, C bp, 0C D. cucurbitae D. dorsalis

82-83

76-78

36-38

72-74

182-18547-48149-151

86-88.5

96-98

129-13138-4098-10037-3896-9735-3656-59

325 dec133-135214137-138170-17387-89181-18369-70168-17163-64

120-122/10 mm

118/0.5 mm123-124/0.2 mm235173-176/30 mm

180-181/30 mm165-167/35 mm160-162/35 mm112-114/0.5 mm129-130/1 mm126-127/20 mm

145/25 mm157-159/10 mm

140/16 mm

310

166-167/25 mm

200/35 mm

136-137/20 mm

87-88/0.2 mm128-129/1.0 mm

>1,0000.03 (F,O)1.0 (F,O)1.0 (F)

10 (F)1,000 (F)

0.1 (F,O)1.0 (F,O)

3,000 (F)10,00010,000

1.0 (F)100 (F)

1,000100 (F)

3,00030 (F,O)

1,000>10,000

1,000 (F)>300

0.3 (F,O)103 (F,O)

>1,0001,000

10 (F,O)0.01 (F,O)

30 (F)10 (F)

>10,00010,000>1,000>1,000>10,00010,000 (F)1,000 (F)

3 (F)103 (F)

>10,0003,0001,0001,000

1.0 (F)1.0 (F)

0.01 (F,O)>1,000>1,000>1,000

1.0 (F)1,000 (F)1,000 (F)3,000

>10,0003,000 (F)

10 (F)>1,000

100 (F)10,0003,000 (F)

3.0 (F)1,000 (F,O)300 (F)

>10,000>10,000>1,0003,000

>1,0003,000

>1,0001.0 (F,O)

>10,000>10,000>10,000>10,000

1,0003.0 (F)

>1,000>1,000

>10,000>10,000>10,000>10,000>10,000>10,000

1,000 (F)3.0 (F)

100 (F)10 (F)

>1,000>1,000

F, males fed at minimum dose; 0, females attempted to oviposit.

producing attraction and a positive feeding response were de-termined (19). The treated filter paper discs were placed on thefloor of 27,000-cm3 (1-ft3) screen cages containing 100 male and25 female flies, and the number of flies attracted to the treatedarea was recorded after 1, 2, 5, 10, and 20 min together withobservations of the behavior of male and female flies (12, 16,19). From 4 to 10 cages of flies of each species were used inrotation to avoid fatiguing the olfactory receptor mechanism.The cages were provided with food and water and were kept inwell-aerated chemical hoods at 69-71°F (21-22°C) to avoid con-

tamination. The data in Table 2 are based on at least four rep-

licate observations of different cages of flies, over a series ofdosages. The preference tests (Table 3) were made by simul-taneous exposure to filter paper treated with equal dosages ofseveral closely related compounds (19). These evaluations were

replicated five times using different cages of flies and random-ized positions of the treated papers.

RESULTSTime Sequence of Raspberry Ketone Response. The re-

sponse of D. cucurbitae to raspberry ketone was remarkablyconstant and was clearly a function of the amount of compoundpresent. When various amounts of raspberry ketone were ex-

posed together in a single cage, the numbers of flies feedingwere directly proportional to the logarithm of the dosage, a

demonstration of the Weber-Fechner law: P = K log S, in whichP = sensation and S = stimulus (12), as shown in Table 2. Thelinearity and reproducibility of response are shown as mean ±

SEM number of flies responding to the various dosages from

IVIIIIVVVIVIIVIIIIXXXIXIIXIIIXIVXVXVIXVIIXVIIIXIXXXXXIXXIIXXIIIXXIVXXVXXVIXXVIIXXVIIIXXIX

XXXIIXXIIXXXIVXXXVXXXVIXXXVIIXX[XVIII

XXIXXLXLIXLIIXLIIIXLIVXLVXLVI

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Table 2. Number of male D. cucurbitae responding tosimultaneous exposure to various concentrations ofraspberry ketone

No., by raspberry ketone concentrationTime, 0.3 1.0min Ag pg 3 tg 10pg 30 Mg

1 0 0 0 1.0±0.9 4.8± 1.32 0 0 2.0±2.3 1.8±1.7 8.6±4.85 0 1.0 3.5 ± 2.0 5.5 ± 2.5 11.4 ± 2.110 0 1.0 2.3 ± 2.0 6.2 ± 2.5 12.2 ± 2.620 0 0.6 1.8 ± 1.5 6.7 ± 3.8 14.4 ± 3.2

Results represent mean ± SEM.

five replicate evaluations. The cumulative response of D. cu-curbitae to raspberry ketone is slower than that of D. dorsalisto methyl eugenol (12) because of the substantially lower vaporpressure of raspberry ketone [bp ca. 400TC at 760 mm (extrap-olated from bp 130-140'C/0.05 mm; ref. (7)] vs. methyl eu-genol (bp 24800 at 760 mm).

Nature of Kairomone Receptors in D. cucurbitae and D.dorsalis Males. The data on the LR for these two species ofDacini to approximately 40 compounds in Table 1 indicate thegreat divergence in species response to simple phenylpropa-noid analogues. D. cucurbitae is extremely responsive to rasp-berry ketone [4-(p-hydroxyphenyl)-2-butanone, II] while D.dorsalis is totally nonresponsive, whereas D. dorsalis is ex-tremely responsive to methyl eugenol (3,4-dimethoxyallylben-zene, I) while D. cucurbitae is nonresponsive. D. dorsalis con-sistently responded to nonpolar 3,4-dimethoxyphenyl compounds(e.g., XI, XVI, XXVI, XXXII, and XLII) at relatively low LRvalues (19) while D. cucurbitae was virtually nonresponsive. Incontrast, D. cucurbitae responded to nonpolar p-hydroxy-phenyl compounds at relatively low LR values (e.g., II, VII,XXII, and XXVIII) while D. dorsalis was nonresponsive. D. cu-curbitae was also responsive to p-methoxyphenyl compounds(e.g., III, VIII, XXIV, XXX, and XL) to which D. dorsalis wasessentially nonresponsive.

Characterization of the Antennal Receptor Site of Male D.cucurbitae. This kairomone receptor is characterized by max-imum complementarity to methyl phloretate (XXVIII; LR, 0.01)and to raspberry ketone (II; LR, 0.03) two naturally occurringphenylpropanoids that have almost identical stereochemistryincluding the key features of (i) a p-hydroxyphenyl, and (ii) aC-O group two atomic diameters from the phenyl ring. Theimportance of these features is shown by the intense odorantproperty of p-hydroxybenzyl acetate (VII; LR, 0.1), which is anisostere of raspberry ketone. Removal of the p-hydroxy groupas in 4-phenyl-2-butanone (V; LR, 10) and benzyl acetate (XIII;LR, 10) decreased receptor affinity by a factor of 1/300 to 1/100. Removal of the C0= group as in 1-(p-hydroxyphe-nyl)butane (VI; LR, 1,000) resulted in very low affinity, sug-gesting that the primary receptor site in D. cucurbitae is thatcomplementary to the aliphatic C=O group.

Position of the C=O group. This is critical for maximum re-ceptor affinity as shown by methyl 3-(p-hydroxyphenyl)pro-panoate (XXVIII; LR, 0.01) (methyl phloretate), methyl p-hy-droxyphenylacetate (XXII; LR, 0.3), and p-hydroxyphenyl pro-panoate (XVII; LR, 30) and by 4-(p-methoxyphenyl)-2-butan-one (II; LR, 1.0), 3-(p-methoxyphenyl)-2-propanone (XV; LR,100), and p-methoxyacetophenone (XIV; LR, 1,000). Comparealso methyl p-methoxybenzoate (XX; LR, 1,000), methyl p-methoxyphenylacetate (XXIV; LR, 3), methyl 4-(p-methoxy-phenyl)propanoate (XXX; LR, 10), and methyl 4-(p-methoxy-phenyl)butanoate (XXXIV, >1,000). Maximum receptor affin-

ity occurs when theC=O group is two atomic diameters removedfrom the phenyl ring (see Table 3, group E).The male D. cucurbitae response by orientation and com-

pulsive feeding was almost identical to the three pairs of ketoneand ester isosteres (II and VII), (III and VII), and (IV and XII)in which the C=O groups have similar steric configurations.Reversal of the C=O group in the two ester pairs-methyl p-hydroxyphenylacetate (XXII; LR, 0.3) vs. p-hydroxybenzyl ace-tate (VII; LR, 0.1) and methyl p-methoxyphenylacetate (XXIV;LR, 3) vs. p-methoxybenzyl acetate (VIII; LR, 1.0)-producedonly a slight decrease in receptor affinity as the configurationsof the C-O groups are very similar (Fig. 1). Some of theseeffects are illustrated by the preference tests (Table 3, group A).From the standpoint of human olfaction, all four substances (II,VII, XXII, and XXVIII) shown in Fig. 1 have similar berry-likeodors.

Effect of hydrophobicity. The receptor affinity in male D.cucurbitae for the relatively hydrophobic carboxylic acid methylesters was from 3-1,000 times greater than that for the morehydrophilic carboxylic acids (compare XIX vs. XX), (XXI vs.XXII), (XXI vs. XXIV), (XXVII vs. XXVIII), (XXIX vs. XXX),(XXXVII vs. XXXVIII), and (XLI vs. XLII).

Receptor complementarity to the p-hydroxyphenyl group. Themale D. cucurbitae exhibited maximum responses to methylphloretate (XXVIII), raspberry ketone (II), and p-hydroxyben-zyl acetate (VII), which have the common feature of a p-hy-droxyphenyl group. The substitution of p-methoxy 'for p-hy-droxy as in HI (anisyl acetone), VIII, and XXX substantiallyreduced receptor affinity. Substitution of p-CH3C(O)O for p-hydroxy as in IV (cue-lure) and XII also reduced receptor af-finity by a factor of 1/30 to 1/10. Therefore, it appears thatdepolarization of the male antennal receptor of D. cucurbitaeis hindered by increasing the lipophilicity of the para substi-tuent [II values: p-OH, -0.67; p-CH30, -0.02; p-CH3C(O)O,

A

B

C

D

k^FIG. 1. Molecular models of strong attractants for D. cucurbitae.

(A) Raspberry ketone (II). (B) Methyl phloretate (XXVIII). (C) p-Hy-droxybenzyl acetate (VII). (D) Methylp-hydroxyphenylacetate (XXII).Note the relative positions of the C=O groups.

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0.31] (20). This conclusion is supported by the preference tests(see Table 3, group D).The receptor in D. cucurbitae is exclusively para oriented.

Unsubstituted 4-phenyl-2-butanone (V; LR, 10) and benzyl ace-

tate (XIII; LR, 100) are moderately strong odorants but p-hy-droxy substitution as in II and VII increased receptor affinity300- to 1,000-fold and p-methoxy substitution (III and VIII) in-creased it 10- to 100-fold. m-Methoxy (IX)-, o-methoxy (X)-, and3,4-dimethoxy (XI)-benzyl acetates were completely inactive.

Preference Tests. Simultaneous exposures to equivalent dosesof three or four closely related odorants in single cages of fruitflies have yielded useful confirmatory information about theodor preferences of D. dorsalis (12, 19). This technique was em-ployed with D. cucurbitae using odorant molecules more or lessclosely related to raspberry ketone (Table 3). The data (groupA) show that D. cucurbitae had a slight preference for raspberryketone (II) over methyl phloretate (XXVIII) and p-hydroxy-benzyl acetate (VII), although both of the latter were able toattract male flies in the presence of raspberry ketone. Similarly(group B), anisyl acetone (I) was selected over p-methoxy-benzyl acetate (VI) and methyl p-methoxyphenylacetate (XXIV).The data for group C demonstrate the specific response of D.cucurbitae to para orientation of the methoxy group of themethoxybenzyl acetates and the complete lack of response with3,4-dimethoxybenzyl acetate. The same set of preference testswith D. dorsalis showed the flies responding only to 3,4-di-methoxybenzyl acetate.The preference tests (group D) comparing raspberry ketone

and its p-alkoxy analogues showed a clearcut preference forraspberry ketone over the p-methoxy derivative (anisyl ace-

tone). The D. cucurbitae were more strongly held by raspberryketone, as shown by the uniformity of response in the five rep-licates (low SEM).

Oviposition Behavior of D. cucurbitae Chemicals that at-tract and arrest male Dacinae also can act as oviposition stim-ulants for female Dacinae. The presence of approximately 25females together with 100 males in the cages used to evaluate

the male response afforded an opportunity to study female be-havior as well. Female D. dorsalis were often observed at-tempting to oviposit on filter paper discs treated with methyleugenol and related 3,4-dimethoxyphenyl attractants (19). WithD. cucurbitae, female oviposition behavior was frequently ob-served on filter paper discs treated with raspberry ketone (II)and with the p-hydroxyphenyl and p-methoxyphenyl com-

pounds HI, VII, VIII, XXII, XXVII, and XXVIII that were alsothe most active attractant/arrestants for the male flies (Table1). Thus, under appropriate conditions, kairomones that are

highly attractive feeding stimulants for male Dacinae also at-tract female flies and stimulate them to oviposit.

Evolutionary Aspects of Male Dacini Antennal ReceptorResponse. Drew and co-workers (5, 9) have shown that the Da-cini can be categorized into two major groups by the male at-traction to either methyl eugenol or raspberry ketone. No spe-

cies responded to both lures and the kairomone responses were

clearly of evolutionary significance in that this "chemotaxon-omy" conforms substantially to morphologically arranged taxa.Both methyl eugenol and raspberry ketone have a common bio-synthetic precursor, cinnamic acid. With the evolutionary de-velopment of oxygenase enzymes in plants, para hydroxylation(e.g., of phenylalanine to tyrosine or of cinnamic acid to p-cou-

maric acid) was a likely first step in kairomone transformation.The para position in the phenyl propanoids is more electro-philic (for CH=CHCOOH, o-, = 0.90 vs. mO= 0. 14; ref. 20)and should therefore be more responsive to nucleophilic attackby the HO'oxygenase moiety. Subsequently, the p-hydroxy-phenylpropanoids were methoxylated to -methoxy and still fur-ther oxygenated to form 3,4-dihydroxy-, 3-methoxy-4-hy-droxy-, and eventually 3,4-dimethoxyphenylpropanoids (21).This suggests that the dominant response of D. cucurbitae andthe preponderant number of related species attracted to rasp-

berry ketone represents the more primitive condition and thatthese species are direct descendants of Dacini whose originalcoevolutionary association could have been with plants con-

taining p-hydroxycinnamic acid. Then, as the secondary plant

Table 3. Preference tests of male D. cucurbitae among groups of related raspberry ketone analogues in single cagesFlies attracted, no.

Group Analogue Dose, /g 1 min 2 min 5 min min 20 min

A 4-HOC6H4CH2CH2C(O)CH3 100 7 ± 3 8 ± 2 10 ± 2 11 ± 3 11 ± 44-HOCCH4CH2CH2C(O)OCH3 100 5 ± 4 5 ± 4 5 ± 4 6 ± 4 5 ± 14-HOC6H4CH20C(O)CH3 100 1 ± 1 2 ± 2 3 ± 3 3 ± 3 3 ± 34-HOC6HACH2C(O)OCH3 100 1 ± 1 1 ± 1 1 ± 1 1 ± 2 0.4 ± 0.5

B 4-CH3OC6H4CH2CH2C(O)CH3 100 4 ± 3 6 ± 3 6 ± 3 7 ± 5 12 ± 64-CH30C6H4CH20C(O)CH3 100 2 ± 2 3 ± 4 3 ± 4 4 ± 4 6 ± 54-CH30C6HACH2C(O)OCH3 100 2 ± 2 2 ± 3 2 ± 3 2 ± 3 3 ± 34-CH3OC6H40C(O)CH2CH3 100 0 0 0 0 0

C 4-CH30C6H4CH20C(O)CH3 100 1 ± 1 1 ± 1 2 ± 2 2 ± 2 5 ± 63-CH30C6H4CH20C(O)CH3 100 0 0 0 0 02-CH3OC6H4CH20C(O)CH3 100 0 0 0 0 03,4-di-CH3OC6H4CH20C(O)CH3 100 0 0 0 0 0

D 4-HOC6H4CH2CH2C(O)CH3 100 8 ± 3 9 ± 2 11 ± 2 11 ± 2 14 ± 34-CH3OC6H4CH2CH2C(O)CH3 100 6 ± 5 7 ± 5 7 ± 6 7 ± 6 8 ± 74-C2H5OC6H4CH2CH2C(O)CH3 100 2 ± 2 2 ± 2 2 ± 2 2 ± 2 2 ± 24-C3H70C6H4CH2CH2C(O)CH3 100 4 ± 3 3 ± 2 3 ± 3 3 ± 2 3 ± 3

E 4-CH30C6H4CH2CH2C(O)CH3 300 3 ± 1 4 ± 1 8 ± 1 9 ± 3 10 ± 24-CH3OC6H4CH2C(O)CH3 300 0 0 0 0 04-CH3OC6H4C(O)CH3 300 0 0 0 0 0

Data are mean ± SEM for five (groups A-D) or four (group E) replicate experiments.

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compounds became more lipophilic through methylation andacetylation and were sequestered as essential oils, the antennalreceptor site was modified to accept more lipophilic molecules.This speculation is supported by the positive feeding responseof D. cucurbitae to p-hydroxycinnamic acid (XXXVII; LR, 1,000)and the much greater sensitivity to the more lipophilic methylester (XXXVII; LR, 3). Subsequent reduction of the C==C bondled to methyl phloretate (XXVIII; LR, 0.01).and ultimately toraspberry ketone (II; LR, 0.03). Small changes in the. male an-tennal receptor site occurred to accommodate the increasingarray of more lipophilic plant essential oils (kairomones) in newlyevolving plants. D. dorsalis and the smaller group of relatedspecies responding to methyl eugenol apparently represent de-scendants of a mutant form whose antennal receptors devel-oped appropriate complementarity to the 3,4-dimethoxyphen-ylpropanoids, thus opening up new ecological niches. There isgood evidence that the primary electronic site in the male D.dorsalis receptor is complementary to the p-methoxy group ofmethyl eugenol [e.g., p-methoxyanisole (LR, 100) vs. m-meth-oxyanisole (LR > 10,000); ref. 12]. This is supported. by therelatively high attractivity of p-methoxypropenylbenzene ortrans-anethole. (LR 10) to D. dorsalis.

Chemical Ecology of Host Selection. The role of the phen-ylpropanoid kairomones in regulation of the behavioral ecologyof the Dacini is complex. Methyl eugenol and raspberry ketonehave been described as parapheromones (10, 22), allelochemicsperhaps related to sex pheromones and promoting intraspecificresponses between male and female. However, in the Dacini,the true sex pheromones are generally produced in rectal glandsof the males and attract virgin females. The sex pheromones ofD. dorsalis and D. cucurbitae act interspecifically in attractingfemales of the other species, indicating a very similar chemicalconstitution (23). The male sex pheromone of D. cucurbitaeconsists of a complex mixture of nitrogen compounds of whichN-3-methylbutylacetamide appears to be the most active inpromoting increased flight activity and searching in the female(24).

It has been suggested that host plant odors act as, rendezvousstimulants-for the Tephritidae to bring the sexes together in theenvironment of suitable host plants (11, 25). Both methyl eu-genol and raspberry ketone act in picogram quantities as at-tractants, arrestants, and compulsive-feeding stimulants for maleDacini (19) and, under some circumstances, as oviposition stim-ulants for female Dacini.The strong odorant activity of phloretic acid and its methyl

ester to D. cucurbitae males, together with the identificationof this acid and its esters in a variety of plants and especiallyin Cucurbita pepo blossom (26), suggests an important role forthese as' kairomones-for host selection.for D. cucurbitae and therelated group of Dacini responding to cue-lure.and Willison'slure (9, 10). At least 10 species of these Dacini have been re-corded as developing in Cucurbitaceae. Therefore, we view thespectrum of secondary plant compounds highly attractive to~maleDacini as kairomones promoting host selection through a com-

plex chemical ecology involving male attraction and compulsivefeeding, liberation of the sex pheromones to attract virgin fe-males, and short-range ovipositional stimulation of the fertil-ized females. The nature of the total kairomone blend and otherfactors such as color,, texture, and nutritional status of the po-tential host plants must also be important.We acknowledge the generous supply of reared oriental fruit flies

and melon flies and cue-lure by the U.S. Department of AgricultureTropical Fruit and Vegetable Research Laboratory, Honolulu, Hawaii.Professor Nelson J. Leonard provided valuable suggestions aboutchemical synthesis and Dr. Hans Hummel participated in some.of theevaluations. The work was supported in part by a grant from the Centerfor Advanced Study, University of Illinois.

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Miyashita, D. H. (1960) Science 131, 1044-1045.4. Alexander, B. N., Beroza, M. O., Steiner, L. F., Miyashita, D.

H. & Mitchell, W C (1962)J. Agr. Food Chem. 10, 270-276.5. Drew, R. A. I. (1974) J. Austr. EntomoL Soc. 13, 267-270.6. Keiser, I., Nakagawa, S., Kobayashi, R. M., Chambers, D. L.,

Urago, T. & Doolittle,.R. E. (1973)J. Econ. Entomol 66, 112-114.7. Schinz, H. & Seidel, C. F. (1961) Helv. Chim. Acta 44, 278.8. Murakami, T. & Tanaka, K. (1972) Tetrahedron Lett. 29, 2965-

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(1979) Proc. NatL Acad. Sci. USA 76, 1561-1565.13. Manitto, P., Monti, D. & Gramatico, P. (1974)J. Chem. Soc. Per-

kin Trans. 1, 1727-1731.14. Friedrich, H. (1976) Lloydia 39, 1-7.15. Geissman, T. H. & Crout, D. H. G. (1969) Organic Chemistry of

Secondary Plant Metabolism (Freeman, Cooper, San Francisco),p. 150.

16. Metcalf,-R. L., Mitchell, W C., Fukuto, T. R. & Metcalf, E. R.(1975) Proc. Natl Acad; Sci. USA 72, 2501-2505.

17. Nago Y., Fujita, E., Kohno, T. & Yagi, .M. (1981) Chem. Pharm.Bu Japan 29, 3202-3207.

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Acad. Sci. USA 78, 4007-4010.20. Hansch, C., Leo, A., Unger, S. H., Kim, K. H., Nikaitani, D. &

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tochemistry, eds. Swain, T., Harborn, J. B. & van Summers, C.F. (Appleton-Century-Crofts, New York), Vol. 12, pp. 91-137.

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Phytochemistry 21, 1935-1937.

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