Pheromonal Communication in the Oriental Fruit Moth and Oriental Fruit Fly

Ritsuo Nishida

Pesticide Research Institute, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

ABSTRACT. The Oriental fruit moth, Grapholitha molesta, and the Oriental fruit fly, Bactrocera dorsalis, infest various kinds of fruits during their larval stage. Males of these insects use volatile sex pheromones to attract the conspecific females during courtship. Male sex pheromone of the Oriental fruit moth secreted from the hairpencil organs is composed of a mixture of ethyl trans-cinamate and methyl epijasmonate and the blend acts as a short-distance attractant for the female. Male sex pheromone of the Oriental fruit fly is secreted from the rectal glands. The pheromonal components, 2-allyl-4,5-dimethoxyphenol and trans-coniferyl alcohol are phenylpropanoids derived from the specific male attractant, methyl eugenol, acquired by foraging from plant sources during adult stage. In both cases, the chemical communication system appears to involve ecological adaptation mechanisms associated with plant secondary metabolites.

Key words: Grapholitha molesta, Bactrocera dorsalis, sex pheromone, ethyl trans-cinamate, epijasmonate

INTRODUCTION

A number of fruit moth and fruit fly species are serious pests infesting deep inside fruits during their larval stages. Both the Oriental fruit moth, Grapholitha molesta (Lepidoptera: Tortricidae), and the Oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae), are destructive pests originated in Asia and distributed in many countries over the world. Although the insect taxa, host fruits, habitat and life history are entirely different to each other, the mating sequence in both cases involves chemical communication with sex pheromones produced by males. Here, I review the pheromonal communication systems of these two important fruit pests and discuss the ecological significance of the pheromonal substances in their life history. The study on the Oriental fruit moth was carried out at New York State Agricultural Experiment Station and Pesticide Research Institute, Kyoto University under a collaboration work with Drs. W.L. Roelofs, T.E. Acree (Cornell University) and T.C. Baker (pres

ent, Iowa State University). The study on the Oriental fruit fly was carried out under a collaboration work with Drs. Tan Keng Hong (Universiti Sains Malaysia), Todd E. Shelly and Kenneth Y. Kaneshiro (University of Hawaii).

THE ORIENTAL FRUIT MOTH

The Oriental fruit moth, G. molesta, is one of the major pests of apples, peaches, plums, pears and other rosaceous fruits. The larvae damage the young twigs, causing them to die in early spring, and the later generations feed inside the fruit rendering them undesirable.

Fig 1. Courtship sequence of the Oriental fruit moth, Grapholitha molesta.

Like most other moth species, females of G. molesta secrete sex pheromone to attract males. The attractant components have been established to be a mixture of Z- and E-8-dodecenyl acetates, Z-8-dodecenol and dodecanol (Roelofs et al., 1969; CardēÜet al., 1979), which are not unusual components for a female sex pheromone of a Lepidoptera. However, the entire sequence of courtship behavior in G. molesta in unique among Lepidoptera in that males attract females after they themselves have been attracted to the vicinity of a female by her sex pheromone as shown in Fig. 1 (Baker and Carde, 1979). Immediately after landing near the calling female [1 - 2], the male turns away and rhythmically extrudes and retracts his abdominal hairpencils, propelling over the female using wind generated by his vibrating wings [3]. The female quickly walks toward the source of the odor and with her head touches the tip of the males abdomen [4], evoking from him a copulatory attemt [5]. The males wing rest on the

top of the females wings [6]. The whole sequence of the courtship behavior provides a rare example of chemical communication between both sexes. Evidently, the male hairpencils contain sex pheromone to attract female moths.

1. Male Hairpencil Pheromone

The hairpencil organs are located between the 7th and 8th abdominal segments of the male moths, and are associated with claspers. Each bundle of the hairpencils is composed of 93 hollow scales which are packed together in special pockets in the abdomen. When extruded, thehairpencils emit a pleasant floral odor, which has appeared to be responsibla for the attraction of females.

The emission of hairpencil volatiles is accompanied with wind puffs created by wing vibration. This anemotactic stimulus appeared to be important under exposure to the hairpencil chemicals. The female moths responded to the chemical stream only when they are calling. Thus, the bioassay was carried out in a windtunnel under a constant wind movements (70 cm/sec) while the female moth were in a calling posture at around sunset time. Females usually walk upwind and then touched the filter paper treated with crude hairpencil extracts.

The hairpencil extract was fractionated by means of gas chromatography (GC). Only a weak activity was recovered from a very volatile fraction, from which ethyl trans-cinnamate (1) was identified. Authentic ethyl trans-cinnamate elicited the same attractant response in females as that evoked by the corresponding GC-fraction and a significant electroantennogram (EAG) response on the female antennae. A synergistic compound with a characteristic floral odor of the hairpencil was isolated from a less volatile GC-fraction, and identified as methyl epijasmonate (2). Although compound 2 was inactive alone, a blend of 1 and 2 exhibited potent attractant activity equivalent to that of the crude hairpencil extract. Thus the male pheromone responsible for attracting females was determined to be a mixture of compounds 1 and 2 (Baker et al., 1982; Nishida et al., 1982). Even though the hairpencil extracts contained R-(-)-mellein (3) in a large quantity, the females did not respond to the compound alon

e. The amounts of compounds 1, 2 and 3 were estimated to be 0.5, 0.01 and 20 ng, respectively.

Fig. 2. Pheromonal components in the male hairpencils of the Oriental fruit moth, Grapholitha molesta. 1: Ethyl trans-cinnamate, 2: (+)-1S,2R-Methyl epijasmonate, 3: (-)-R-Mellein

2. Male-to-Male Interaction

The most abundant hairpencil volatile mellein (3) showed no significant effect of female attraction, and also it was electrophysiollogically inactive to the female antennae. However, thw EAG assay showed a significant response to the male antennae, suggesting its role in the male-to-male interaction (Nishida et al., 1985). In order to examine the possible function of mellein in males, the authentic (-)-R-mellein was tested as a wind puff directed toward males that were resting on a screen of a male cage during mating period. Males walked away or flew from the chemical source as soon as the air puff was blown to the male head. No such efect was observed towards females (10 - 1000 ng of 3 applied to a piece of filter paper in a glass pipet used for EAG assay). The crude hairpencil extracts sometimes induced repulsive behavior as well as mellein. The crude extract, however, more often attracted male moths frequently triggering hairpencilling behavior, which suggested a complexity of its role

in the interactions between males.

3. Acquisition of Pheromone Components

Since both cinnamic jasmonic esters (1 and 2) and their derivatives are known plant constituents, it is possible that the male moths acquire the components from their larval diet. Preliminary studies indicated that hairpencils from the male Oriental fruit moth reared on an artificial diet do not possess the characteristic odor of hairpencils. The hairpencil extract did not contain any detectable amount of 1 and 3 on GC, whereas the major component, mellein (3), was present at a normal level. Varieties of the host fruits have been analyzed on GC, but the contents of 1 - 3 were below detectable levels. The insect might biosynthesize these compounds from unknown precursors in the host fruits possibly in the assistance of some symbiotic microorganisms.

Onthe other hand, the adult males have been found to have an ability to sequester one of the hairpencil components (1) in the hairpencils. The adult moth (both sexes) sometimes visit injured fruits to ingest the juice. Fruit juice or the fermentation products have been used to attract the adult moths to monitoring traps ((Ueno et al., 1960). Rotting fruits produce characteristic flavor components that differ from fresh fruits; ethyl trans-cinnamate (1) was detected from rotting Japanese pear that actually attracted the mohs in the orchard (Nishida et al., 1985). The tracer analysis using 14C-labeled and pentadeuteroethyl trans-cinnamate proved the incorporation of 1 into the hairpencil organs by direct ingestion with sucrose solution during the adult stage. By contrast, trideuteromethyl trideuteroepijasmonate was not incorporated into the hairpencils, when supplied with sucrose solution. Ethyl trans-cinnamate is highly volatile and the moth can have a chance to sequester an additional qu

antity by foraging from rotten fruits together with nutrients. Since the males mate several times in their lifetime and the enrichment of hairpencil pheromone would ensure the multiple copulations, acquisition of additional 1 during adult stage may be of biological significance in regadr to mating competetion.

THE ORIENTAL FRUIT FLY

Males of many fruit fly species in the subfamily Dacinae are strongly attracted to synthetic aromatic compounds, some of which occur as essential oil components in various plants (Chambers, 1977; Fletcher, 1987). Among the economically important species in the Asian Pacific area, methyl eugenol [3,4-dimethoxyallylbenzene (4)] acts as a highly potent attractant for males of the Oriental fruit fly, Bactrocera dorsalis, and cue-lure and its deacetyl derivative [4-(4-hydroxyphenyl)-2-butanone] act as specific attractants for the melon fly, B. cucurbitae. The fruit flies attracted to these chemical sources are exclusively males, and females usually do not respond to the lures. The flies attracted to the chemical sources voraciously consume the intact lure chemicals. These attractants have been successfully used as the mass trapping agents and population-monitoring bates in the eradication program for these pests (Chambers, 1977). However, the biological meaning of such specific affinities of th

e male Bactrocera flies to the lure chemicals was not fully understood. We have examined the ecological roles of male attractant in the Oriental fruit fly complex species (Bactrocera dorsalis and B. papayae), and demonstrated that lure compound served as a precursor for male sex pheromone as well as allomone (Tan and Nishida, 1996; Nishida and Shelly, unpublished).

1. Rectal Accumulation of Phenylpropanoids

Methyl eugenol is a phenylpropanoid compound widely distributed in plants, and B. dorsalis males frequently congregate on the lure plants such as Cassia fistula and Ocimum sanctum, and persistantly lick the attractant sources (Kawano et al., 1968; Shah and Patel, 1976). The foraging behavior can be seen in the early daytime and all the flies fly away before sunset. Wild males were collected at various field sites in Malaysia and Hawaii and the volatile components in the tissue were examined by means of chromatography and spectrometry. Two major volatile substances 2-allyl-4,5-dimethoxyphenol (5) and trans-coniferyl alcohol [4-(3-hydroxyl-E-propenyl)-2-methoxyphenol (6)] were found in unusually large quantities. In contrast, extracts of the sexually mature males from a laboratory-reared culture (fed with water and a honey-teast mixture during adult stage) entirely lacked these compounds. These compounds are oxygenated derivatives of methyl eugenol, suggesting the compounds to be direct meta

bolites of 4 (Nishida et al., 1998a).

When the laboratory-reared males (8 days after adult eclosion) were fed individually with methyl eugenol for several minutes, the compound was quickly consumed, and 5 and 6 were markedly built up in their tissue. Thus the wild males must have consumed 4 from certain plant sources during foraging, and sequester the metabolites (5 and 6) in their body in varying quantities. The quantitative variation seemed to be dependent upon the availability of 4 in their natural habitat and also physiological age of flies (Nishida et al., 1988a).

Fig. 3. Phenylpropanoid analogs associated with the life history of Bactrocera dorsalis [4: methyl eugenol, 5: 2-allyl-4,5-dimethoxyphenol, 6: trans-coniferyl alcohol]

Dissection of the male insect body revealed that a large portion of the metabolites 5 and 6 accumulated in the rectal glands (Nishida et al., 1988a). Some of the B. papayae specimen from 1 day post feeding with 4 also possessed analogous compound 7, which was identified as cis-3,4-dimethoxycinnamyl alcohol (Nishida et al., 1988b). When the males were fed with a large excess of 4, the intact 4 was found in the body tissues but not in the rectal glands. Methyl eugenol (4) seems to be quickly metabolized to its oxidized forms (5,6 and 7) in the body tissue and selectively incorporated in the rectal sacs. Besides these phenylpropanoids, N-3-methylbutyl acetamide was detected as one of the prominent volatiles in the gland extracts of D. dorsalis males regardless of feeding or unfeeding with 4.

2. Pheromonal Function of Rectal Phenylpropanoids

D. dorsalis males produce a smoke-like substance from the rectal glands, which has been shown to be attractive to virgin females (Ohinata et al., 1982). The volatiles from a chamber containing D. dorsalis males which had fed with methyl eugenol were collected on a short column of porapak-Q. The trap was replaced every 2 hours starting from noon until midnight. 2-Allyl-4,5-dimethoxyphenol (5) was recovered from the aeration extract only during the time around sunset (17:00-19:00 pm) which coincides with the period when D. dorsalis males congregate and attract females. This evidence strongly suggests a possible role of methyl eugenol as a pheromone precursor in a courtship behavior, including male-to-male interaction (Nishida et al., 1988a; Nishida and Fukami, 1990).

It has been clearly shown that males fed on methyl eugenol (4) were more successful in mating competetion with unfed males (Shelly and Dewire, 1994; Tan and Nishida, 1996). Many of the unmated females were observed chasing males which had been fed on 4, sometimes extruding the ovipositor directly toward the male. Such behavior was seldom seen toward the unfed males. Since copulation often occurred immediately after such females action, this behavior was suggested to be an acceptance response of the female.

Females were found to become very sensitive to the male rectal substances (5 + 6) sometime after sunset, even though mating usually starts much earlier than that period. Females were attracted to filter paper discs treated with an artificial mixture of 5 and 6, and licked the chemical source, frequently accompanied by ovipositor-extruding behavior toward the sample disc. Rectal metabolites 5 and 6 thus appeared to act as a short-range attractant and arrestant to elicit female acceptance of male courtship (Tan and Nishida, 1996; Nishida abd Shelly, unpub.).

Since a mixture of 5 and 6 also attract unfed males, inducing compulsive feeding behavior at the point where the fed males had secreted the rectal substance, the behavior might be associated also with male-to-male competition in the sexual process.

3. Allomonal Function of Phenylpropanoids

Although the quantities of the phenylpropanoids accumulated in wild B. dorsalis tissue varied individually, some of the flies contained over 20 µg of 5, the quantities of which seemed to be unusually large for a sex pheromone compared to such as moth pheromones. The potent phagostimulant activity of phenylpropanoids was suggested to be associated also with chemical defense mechanism against predators by sequestering the distasteful or toxic principles in the insect bodies (Nishida et al., 1988a). An allomonal effect of the metabolites has been shown by deterrence bioassay using the Japanese tree sparrow, Passer montanus. Deterrent effect of compounds 4, 5 and 6 were examined in the rice grain feeding test (Nishida and Fukami, 1990). Compound 5 was shown to act as a potent deterrent. Compound 4 was less active than 5, and 6 was inactive at the given dossage. Conversion of methyl eugenol to more potent oxidized form 5 might be adaptive in this insect. Although coniferyl alcohol (6) was inacti

ve to sparrows, the compound and its analogs have been shown to exhibit potent deterrent activity against other predatory birds (Jakubas et al., 1992).

DISCUSSION

Although volatile chemicals identified from male scent organs or from other specialized scales have been described in many Lepidoptera, the pheromonal functions of those compounds have been elucidated only in rare instances (Birch et al., 1990). The overt movement of females toward hairpencil-displaying males in the Oriental fruit moth provided a opportunity to define a courtship pheromone that attract females. This suggests the hairpencil volatiles produced by male lepidopterous species primarily to act as sex recognition pheromone, even though the pheromone exert a minimal observable behavior on females in most cases.

Male rectal gland complex of the dacine fruit flies was suspected to be the source of an olfactory pheromone without clear behavioral proof in the courtship sequence (Schultz and Boush, 1971; Kobayashi et al., 1978). The specific affinity of the male Oriental fruit fly to methyl eugenol has provided us a clue for the consequent behavioral evidence that the males utilize phenylpropanoid metabolites accumulates in the rectal glands as a sex pheromone to attract females (Tan and Nishida, 1996; Nishida and Shelly, unpublished).

In both instances, the insects (G. molesta and B. dorsalis) mate at around sunset time. Recognition of males by females under dusk condition appeared to be effectively assisted by those short-distance pheromone from the specialized organs. Prior to mating, males of both moth and ruit fly often show gregarious behavior in a restricted area in the natural habitat; particularly in the latter case forming a lek (Shelly and Kaneshiro, 1991). The volatile substances disseminatesd from the male secretory organs may function as a pheromone not only toward females but toward males in the rigorous interactions between males before courtship. The rectal phenylpropanoid produced by methyl eugenol-fed B. dorsalis males may trigger the lek formation, since the compounds strongly attract and arrest males as well. In the case of G. molesta, the hairpencil substance sometimes attracts males, whereas the most prominent constituent mellein repels males. Aggregation and repulsion within males prior to matin

g suggest an involvement of those olfactory cues in male-to-male competetions.

Male pheromone components of the Oriental fruit moth are suggested to be originated from the larval host plants. Above all, jasmonic esters have recently been shown to be plant hormone with various physiological functions (Gundlach et al., 1992). Thus the females preference of males might be influenced by the physiological condition of the host fruits on which the males feed during the larval stage. In addition, males were shown to have an ability to sequester a phenylpropanoid component, ethyl trans-cinnamate, during adult stage. This can be contrasted to the acquisition of methyl eugenol by the Oriental fruit fly males, in that the males obtain the phenylpropanoid components not from the host fruits during larval stages but pharmacophagously from non-host plant sources during adult stage. A large portion of the natural population of the Oriental fruit fly has been shown to store considerably large quantities of phenylpropanoids 4 and 5, suggesting its secondly role in deterring vertebr

ate predation (Nishida and Fukami, 1990; Tan and Nishida, 1996). Therefore, the female preference for males with phenylpropanoids might have been adaptively developed in conjunction with a defensive mechanism in this fruit fly species. In the other context, uses of these male pheromones to entice conspecific females may have evolved through sexual selection, particularly female preference for males scented with fragrances derived from plants, reflecting a runaway process.

The male sex pheromones of both Oriental fruit moth and Oriental fruit fly may not be directly applicable as luring agents to the control programs, since the compounds are effective only as short-distance female attractants together with other sensory modalities. Interestingly, both males and females of the Oriental fruit moth are strongly attracted to certain kinds of fermented fruit juice which often contain ethyl trans-cinnamate and other related derivatives (active principles not known); the fermentation products were used for a monitoring and mass trapping agent for the moth in the pest before the female sex pheromone trap was introduced (Ueno et al., 1960). Strong affinity to the adult-food flavors might be involved in the association with the moth hairpencil pheromone as seen in the pharmacophagous interaction between a danaine butterfly and pyrrolizidine alkaloids (Meinwald et al., 1968; Bopprēč 1986). In the sterile insect technique of the Oriental fruit fly, the mating frequency

of mass reared sterile males may be enhanced by pre-exposure to methyl eugenol. Thus it could effectively achieve the desired eradication program with lower number of sterile males (Tan and Nishida, 1996). Further investigation is necessary to understand the importance of plant secondary substances in the life history of these fruit pests.

Acknowledgments. I wish to thank Drs. Thomas C. Baker, Wendel L. Roelofs and T.E. Acree for studies on the Oriental fruit moth, and Drs. Keng Hong Tan, Todd E. Shelly and Kenneth Y. Kaneshiro for studies on the Oriental fruit fly.

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