Mutual Reproductive Benefits Between a Wild Orchid, Bulbophyllum patens, and Bactrocera Fruit Flies via a Floral Synomone

The solitary flower of Bulbophyllum patens selectively attracts male fruit flies of several Bactrocera species with a specific fragrance in the rain forest of Malaysia. It temporarily traps flies between its hinged see-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 (methyl eugenol and raspberry ketone) and shows potency to attract a wide range of fruit fly species (B. carambolae, B. caudata, B. cucurbitae, B. tau, and B. umbrosa). A fruit fly visitor is rewarded by feeding on zingerone, and in 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 in the pheromone gland to attract conspecific females. Males of B. papayae, however, convert zingerone to zingerol in the crop. The latter compound is stored in the rectal gland and subsequently released to attract females. This provides a rare example of a mutualistic interaction between insects and plants via a floral synomone, in which both organisms obtain advantages directly benefiting their reproductive systems.     

Synomone Or Kairomone? – <Emphasis Type="Italic">Bulbophyllum Apertum</Emphasis> Flower Releases Raspberry Ketone To Attract <Emphasis Type="Italic">Bactrocera</Emphasis> Fruit Flies

Bulbophyllum apertum flower (Orchidaceae) releases raspberry ketone (RK) in its fragrance, which attracts males of several fruit fly species belonging to the genus Bactrocera. Besides RK as a major component, the flower contains smaller amounts of 4-(4-hydroxylphenyl)-2-butanol, plus two minor volatile components, veratryl alcohol and vanillyl alcohol. Within the flower, the lip (labellum) had the highest concentration of RK with much smaller quantities present in petals; other flower parts had no detectable RK. Male fruit flies attracted to the flower belong to RK-sensitive species – such as Bactrocera albistragata, B. caudatus, B. cucurbitae (melon fly), and B. tau. Removal and attachment of the pollinarium to a flys thoracic dorsum occurred when a male of B. albistragata was toppled into the floral column cavity, due to an imbalance caused by it shifting its body weight while feeding on the see-saw lip, and then freeing itself after being momentarily trapped between the lip and column. During this process, the stiff hamulus (the pollinia stalk protruding prominently towards the lip) acted as a crowbar when it was brushed downwards by the toppled fly and lifted the pollinia out of the anther. If the fly was big or long for the small triangular lip, it would not be toppled into the column cavity and would just walk across the column, during which time the pollinarium could be accidentally removed by the flys leg, resulting in a failed transport of the pollinarium. This suggests an unstable situation, where the orchid relies only on a particular pollinator species in the complex ecosystem where many RK-sensitive species inhabit.Wild males of B. caudatus (most common visitors) captured on Bulbophyllum apertum flowers were found to sequester RK in their bodies as a potential pheromonal and allomonal ingredient. Thus, RK can act either as a floral synomone (pollinarium transported) or kairomone (accidental removal of pollinarium leading to total pollen wastage), depending on the body size of the male fruit flies visiting the flowers.     

Floral Phenylpropanoid Cocktail and Architecture of Bulbophyllum vinaceum Orchid in Attracting Fruit Flies for Pollination

It is widely believed that most orchid flowers attract insects by using deception or chemical rewards in the form of nectar. Flowers of Bulbophyllum vinaceum produce a large array of phenylpropanoids that lure tephritid fruit fly males and also act as floral reward, which the flies subsequently convert to pheromone components. The major floral volatile components identified are methyl eugenol (ME), trans-coniferyl alcohol (CF), 2-allyl-4,5-dimethoxphenol (DMP), and trans-3,4-dimethoxycinnamyl acetate, whereas the minor components are eugenol, euasarone, trans-3,4-dimethoxy cinnamyl alcohol, and cis-coniferyl alcohol. Among the various floral parts, the lip (which is held in a closed position up against the sexual organs) has the highest concentration of the major compounds. An attracted male fly normally lands on one of the petals before climbing up onto and forcing the “spring loaded” floral lip into the open position, hence exposing the floral sexual organs. The architecture and location of chemical attractants of the lip compel the fly to align itself along the lip’s longitudinal axis in a precise manner. As the fly laps up the compounds and moves towards the base of the lip, it passes the point of imbalance causing the lip to spring back to its normal closed position. The fly is catapulted headfirst into the column cavity, and its dorsum strikes the protruding sticky base of the hamulus and adheres to it. The momentum of the fly and the structural morphology of the long stiff hamulus act to pry out the pollinia from its anther cover. Hence, the pollinarium (pollinia + hamulus) is detached from the flower and adhered to the fly’s dorsum. In this unique mutualistic association, both species receive direct reproductive benefits—the flower’s pollinarium is transported for cross pollination, and the fly is offered a bouquet of phenylpropanoids (synomone) that it consumes, converts, and/or sequesters as sex pheromonal components, thus enhancing sexual attraction and mating success.     

Relationship of sexual maturation rate to response of oriental fruit fly strains (Diptera: Tephritidae) to methyl eugenol

Laboratory-reared and wild, fruit-reared adults of the Oriental fruit fly,Dacus dorsalis Hendel, were tested for response to methyl eugenol at various ages. Virgin laboratory (2, 4, 6, 8, and 10 days old) and wild (7, 11, 15, 19, and 23 days old) flies were released into an outdoor field cage and trapped over a two-day period. Response of males increased with age as ca. 32% and 22% of laboratory and wild males responded at 2 and 7 days of age, respectively, while ca. 93% of both strains responded at 10 and 23 days of age, respectively. These correspond approximately to the ages at which they reach sexual maturity. Female response did not increase with age and fluctuated between 15% and 29% for the laboratory strain and 10% and 45% for the wild strain. The age-related response profiles, when integrated with sexual maturation curves, indicate that one of the major reasons the male-annihilation technique is effective is because methyl eugenol is able to attract 40–50% of male flies prior to the onset of sexual maturation.     

Behavioral responses of male and female Mexican fruit flies,Anastrepha ludens,to male-produced chemicals in laboratory experiments

The behavioral responses of male and female Mexican fruit flies elicited by male abdominal extracts were measured in laboratory cages where pheromone was applied to the undersides of some leaves on a treated tree but to none of the leaves on a control tree. After arrival to the treated tree, females came directly to pheromone sources. Females on the treated tree visited leaves and fought other females at higher rates than on the control tree. Females stayed on treated leaves and trees longer than on control leaves and trees. In separate experiments, the number of males on pheromone-treated trees and leaves was higher than on controls, but other behavior was unchanged. The results indicate that the pheromone stimulates a complex of behavior involved in the mating ecology of the species.     

Biology of pheromone release by male caribbean fruit flies,Anastrepha suspensa (Diptera: Tephritidae)

Males of the Caribbean fruit fly,Anastrepha suspensa (Loew), typically form leks and attract females by releasing a multicomponent volatile pheromone. Previous reports have identified two nine-carbon alcohols, three lactones, a sesquiterpene, and a monoterpene in the volatiles. The present report is a study of the physiology of male pheromone release and of ecological and social interactions that influence pheromone release by laboratoryreared flies. Volatiles released by males were trapped on Tenax, eluted, separated, and quantitatively measured by gas chromatography. Experiments showed that the volatiles were primarily released from mouth and anus. Sealing the anal opening or the mouth with melted beeswax resulted in up to 40% or greater reduction in most components, and sealing both mouth and anus further reduced release of volatiles, but some volatiles are possibly still released directly from the cuticle. An anal pouch of everted tissue played a major role as a large evaporative surface for release of some of the volatile components. Male flies entrained to a 1410 light-dark cycle showed a peak release of volatiles at 11–12 hr into the photophase, but smaller quantities of the same volatiles were released over a broad period during the daylight hours. Laboratory-reared males peaked in pheromone release at 7–10 days and production and release continued through 35 days of age. Single males released significantly more of all components measured than did groups of males. The reduction by aggregations of males may be related to lekking behavior in this fruit fly. The pheromone probably serves to attract females to a lek site, but additional parameters are likely to enter into the choice of male made by the arriving female.This is University of Florida Agricultural Experiment Station Journal Series No. 9589.     

Behavioral responses of female Mexican fruit flies,Anastrepha ludens,to components of male-produced sex pheromone

The behavioral responses of virgin female Mexican fruit flies elicited by components and combinations of the components of male-produced pheromone were measured in a laboratory wind-tunnel bioassay where test chemicals were applied to the undersides of some leaves on a treated tree but to none of the leaves of a control tree. Only treatments containing at least (Z)-3-nonenol and/or (Z,Z)-3,6-nonadienol in combination with (S,S)-(–)-epianastrephin elicited strong behavioral responses. Responses included attraction to the vicinity of the pheromone but not to point sources, increased searching rate, changes in searching strategy, and agonism. The results support a model of pheromone-component function in which components act as a unit to stimulate all behaviors of the pheromone-mediated behavioral repertoire.Diptera: Tephritidae.     

Attraction of female mediterranean fruit flies to the five major components of male-produced pheromone in a laboratory flight tunnel

Attraction and pheromonal activity of five major identified components of the male-produced sex pheromone of the Mediterranean fruit flyCeratitis capitata to virgin laboratory-reared females was assessed in a laboratory flight tunnel. Dual-choice competitive assays were run to establish a baseline response of virgin females to live male pheromone, individual components, and an ensemble of all five compounds alone (air control) and competitively against one another. Approximately 50% of the females released in the tunnel were captured on leaf models emitting pheromonal odors from five live males. Over 37% of released females responded to an ensemble of five major identified components presented in individual capillaries. Response of females to individual components was less than 10%. Competitive assays showed the live male-produced pheromone to be more attractive than either the five major component ensemble (FMCE) or individual components. Further research is likely to identify other male-produced compounds with pheromonal activity that could improve development of a pheromone-based trap for monitoring Mediterranean fruit fly populations.     

Isolation,identification, and synthesis of male-produced sex pheromone of papaya fruit fly,Toxotrypana curvicauda Gerstaecker (Diptera: Tephritidae)

A male-produced sex pheromone of the papaya fruit fly,Toxotrypana curvicauda Gerstaecker, was isolated from volatiles collected from air passed over calling males and was identified as 2-methyl-6-vinylpyrazine by comparative gas-liquid chromatographic and spectroscopic evidence. Synthetic 2-methyl-6-vinylpyrazine elicited typical pheromonal responses from unmated mature female flies such as walking, running, and flying in an arena bioassay; flying upwind with a zigzag flight pattern; and hovering in the pheromone plume in a wind-tunnel bioassay. These responses were similar quantitatively and qualitatively to responses to naturally occurring pheromone from calling male papaya fruit flies.This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or the recommendation for its use by USDA.     

Attraction of Female Oriental Fruit Fly, Bactrocera dorsalis, to Volatile Semiochemicals from Leaves and Extracts of a Nonhost Plant, Panax (Polyscias guilfoylei) in Laboratory and Olfactometer Assays

Fresh whole leaves and solvent–water leaf extracts of the hedgerow plant panax, Polyscias guilfoylei (Bull), were tested for their attractiveness to male and female Oriental fruit flies, Bactrocera dorsalis, in laboratory flight tunnel and cage olfactometer bioassays. Fresh mature whole panax leaves were found to be attractive to mated female oriental fruit flies in the flight tunnel. Response of males and virgin females was low and in most instances not significantly different from controls. Attraction of mated female flies to the layers resulting from a methylene chloride–water partition or a hexane–water partition of freshly ground leaves using small McPhail traps was greatest in the methylene chloride fraction. When methylene chloride and water layers were tested competitively in a multiple-choice rotating olfactometer, the methylene chloride fraction was more attractive. Tests involving the methylene chloride–water interface (an emulsion of the two partitioned layers) with and without a standard attractant NuLure, showed the emulsion layer to be significantly more attractive than the other fractions or NuLure. In outdoor cage olfactometer assays of methylene chloride and water fractions, activity was greatest in the methylene chloride fraction. The results suggests that volatile semiochemicals from this nonhost plant are attractive to mated female Oriental fruit flies. The results are discussed in relation to the chemical ecology of B. dorsalis and the potential use of this nonhost plant for detection and control of female Oriental fruit flies in the field.     

Partial characterization and HPLC isolation of bacteria-produced attractants for the Mexican fruit fly,Anastrepha ludens

Methods were developed to collect and isolate volatile chemicals produced by aStaphylococcus bacterium in tryptic soy culture that are attractive to protein-hungry adult Mexican fruit flies. Centrifugation of bacteria culture yielded a slightly attractive pellet containing most of the bacteria cells and a highly attractive supernatant. Supernatant filtered to remove the remaining bacteria was as attractive as the unfiltered supernatant. Filtrate at pH 7 and above was much more attractive than filtrate at pH 5 and below. Most of the attractiveness was retained on strong cation exchange media under acidic conditions and eluted with base. Attractive principles could not be trapped on adsorbents such as Porapak Q or extracted with organic solvents from aqueous preparations, but they were easily collected by headspace sweeping with steam. The attractive components were efficiently concentrated by rotary evaporation of steam distillate at pH 5, but at higher pH much of the attractiveness distilled. A reverse-phase HPLC method using a negative counter-ion was developed to separate and collect attractive components of concentrated steam distillate. Attractive fractions collected using this method were concentrated and injected onto silica HPLC. Activity eluted from silica in two distinct bands. Results suggest that the most attractive components of the bacterial odor are highly polar, low-molecular-weight amines.Diptera: Tephritidae.     

Attraction of both sexes of Mexican fruit fly,Anastrepha ludens,to a mixture of ammonia,methylamine, and putrescine

Eight chemicals were evaluated in laboratory experiments as attractants for sugar-fed adult Mexican fruit flies. Ammonium bicarbonate, methylamine HCl, ethanolamine, pyrrolidine, putrescine, and monomethyl succinate were slightly attractive when tested singly. A mixture containing all eight chemicals was much more attractive than any of the individual chemicals. Through a series of experiments, a mixture of three of the chemicals was found that was at least as attractive as the original eight-component mixture. The final mixture consisted of ammonium bicarbonate, methylamine HCl, and putrescine in a 10101 ratio. Ratios were less important than actual concentrations of individual components over the range of component concentrations tested. The three-component mixture was equally attractive to male and female flies over at least a 1000-fold range of concentrations and was slightly more attractive thanTorula yeast over the upper 10-fold range in competing McPhail traps in a greenhouse flight chamber.     

1,5,7-Trioxaspiro[5.5]undecane,a pheromone analog with high biological activity for the olive fruit fly,Dacus oleae

l,5,7-Trioxaspiro[5.5]undecane, an analog of the major sex pheromone (olean) of the olive fruit fly, was synthesized via two different routes and tested for biological activity under laboratory and field conditions. In laboratory tests, its activity was comparable to that of olean, especially when a stabilizer or a high concentration was used. In field tests, its activity reached the level of olean only when a stabilizer and an open-type dispenser, which allows high rates of evaporation, were used. The residual activity of the analog dispensers did not exceed two weeks both under laboratory and field conditions compared to over four months for olean.     

Synthesis of attractants for oriental fruit flyDacus dorsalis Hendel using a catalytic organocopper coupling reaction

A general synthetic approach to various catechol derivatives was developed using a copper-catalyzed cross-coupling reaction of 1,2-dimethoxy-4-brornomethyl, 1-ethoxy-2-methoxy-4-bromomethyl- and 2-ethoxy-1-methoxy-4-bromomethylbenzenes with Grignard reagents. Dilithium tetrachlorocuprate was an acceptable catalyst in the dimethoxy series, whereas copper(I) iodide in THF-HMPA was a superior catalyst in all cases due to decreased side reactions, i.e., reduction and reductive coupling. Methyl-substituted analogs of methyl eugenol, a potent attractant of Oriental fruit fly,Dacus dorsalis Hendel, were synthesized by this method and evaluated for attractancy in field tests.On leave from the Institute of Organic Chemistry of the Armenian Academy of Sciences, Yerevan, Armenia; author to whom correspondence should be addressed.Presented at the 204th ACS National Meeting, Washington, D.C., 1992. This paper reports the results of research only. Mention of a proprietory product does not constitute an endorsement of this product by the USDA.     

A four-component attractant for the mexican fruit fly,Anastrepha ludens (Diptera: Tephritidae), from host fruit

Sixteen chemicals found in fermented chapote fruit odor were evaluated as attractants for hungry adult Mexican fruit flies. Ethyl octanoate, ethyl benzoate, terpinyl acetate, ethyl salicylate, and (–)--copaene proved slightly attractive. Several of the chemicals also were tested for their ability to increase the attractiveness of the previously developed chapote-derived attractant (CEH) consisting of 1,8-cineole, ethyl hexanoate, and hexanol. Combinations containing CEH with ethyl octanoate, ethyl benzoate, 4-terpineol, (–)--cubebene, or-terpineol were significantly more attractive than CEH alone. The two most attractive four-component combinations were ethyl octanoate with CEH (CEHO) and ethyl benzoate with CEH. No combinations containing greater numbers of chemicals were significantly more attractive than CEHO. Therefore, CEHO was selected for further study in this paper. Of CEHO component ratios that were tested, the most attractive was 1011100 for the chemicals 1,8-cineole, ethyl hexanoate, hexanol, and ethyl octanoate, respectively. Formulations of CEHO into rubber septa and polyvinyl chloride (PVC) were aged 0–15 days and tested againstTorula yeast in competing McPhail traps in a flight chamber. Summed over all lure ages, rubber septa and PVC dispensers, respectively, were 1.2 and 1.5 times more attractive thanTorula yeast. PVC dispensers aged 10–15 days were approximately 2.1 times more attractive thanTorula yeast.     

Electroantennogram responses of the mediterranean fruit fly,Ceratitis capitata,to a spectrum of plant volatiles

Electroantennograms (EAGs) were recorded from unmated, laboratory-reared, male and femaleCeratitis capitata (medfly) in response to a range of C₁ and C₂ to C₁₂ carbon chain-length aliphatic alcohols, aldehydes, acetates, and acids, and lactones, some of which are known volatiles from leaves and fruits. A large degree of EAG response uniformity between the sexes was observed, with only eight of the 70 compounds tested eliciting significantly larger amplitude EAG responses from female than male antennae. In general, for the five functional-group series tested, aldehydes and alcohols elicited greater responses than acetates, lactones, and acids. The unsaturated alcohols, aldehydes, acetates, and acids elicited equal or larger amplitude EAG responses than their comparable saturated compounds. For four of the functional-group series tested, the EAG response amplitude was significantly greater for a particular carbon chain length, with responsiveness to primary alcohols and aldehydes peaking at C₆, acids peaking at C_5–6, and acetates peaking at both C₅ and C₈. The EAG responses to both the 2- and 3-position monoenic alcohols peaked at C₆ and C₈, while the secondary alcohols peaked at C₇. The greatest EAG responses of all compounds tested were elicited by monoenic C₆ alcohols and aldehydes that are constituents of the general green-leaf odor that emanates from most plants. The potential adaptive benefit of selective sensitivity to green-leaf volatiles is discussed in regard to foraging behavior of medflies.     

Electroantennographic and coupled gas chromatographic-electroantennographic responses of the mediterranean fruit fly,Ceratitis capitata,to male-produced volatiles and mango odor

We have identified five compounds from the headspace of calling male Mediterranean fruit flies (medfly),Ceratitis capitata (Wiedemann), and three compounds from the headspace of ripe mango (Mangifera indica L). using coupled gas chromatographic-electroantennographic (GC-EAG) recordings, coupled gas chromatographic-mass spectrometric (GC-MS) analysis, and electroantennographic (EAG) assays of standards. The male-produced volatiles eliciting responses from female antennae were ethyl-(E)-3-octenoate, geranyl acetate, (E,E)--farnesene, linalool, and indole. An EAG dose-response test of linalool enantiomers and indole with female medfly antennae showed relatively strong EAG activities, but no significant difference between (R)-(-)-linalool and (S)-(+)-linalool. The three mango volatiles were identified as (1S)-(-)--pinene, ethyl octanoate, and-caryophyllene. In addition, a strong antennal response was recorded from a contaminant,-copaene, present in a commercial sample of-caryophyllene. The EAG response amplitudes from both male and female antennae to the above three mango volatiles were significantly greater than to a hexanol control. For both male and female medfly antennae, the greatest EAG responses were elicited by-caryophyllene followed by ethyl octanoate. The mean EAG responses of female antennae to-caryophyllene and (1S)-(-)--pinene were significantly greater than those of male antennae.     

Characterization of laurel fruit oil from Madeira Island,Portugal

The fixed oil extracted from Laurus spp. fruit from Madeira Island, Portugal, is used in local traditional medicine for a wide variety of health complaints. Physical properties, density and refractive index, as well as the TAG FA composition, sterols, and waxes were determined. The oil was found to have an unusually high content of volatiles (ca. 10%), with trans-ocimene and germacrene D predominating. Oleic (30%) and linoleic (20%) acids were the main unsaturated FA, whereas lauric (18%) and palmitic (up to 22.5%) acids were the main saturated FA in the neutral lipid fraction. The oil had a sterol content on the same order as olive oil, with β-sitosterol (84%) predominating. Two sesquiterpene lactones, dehydrocostuslactone and costunolide, accounted for 5% of the overall composition. Madeira laurel oil is not currently used as an edible oil because of its very strong flavor. Its claimed medicinal properties have not yet been validated, and this is the first report on the characterization of the commercial product.     

Hairpencil pheromone components of male oriental fruit moths,Grapholitha molesta

MaleGrapholitha molesta hairpencil components are ethyltrans-cinnamate, mellein, methyl jasmonate, and methyl 2-epijasmonate. The natural behavioral effect elicited by hairpencil-displaying males during courtship in attracting sex-pheromone-releasing females from several centimeters away can be duplicated by mixtures of ethyltrans-cinnamate in various combinations with the other components.Lepidoptera: Tortricidae.Supported in part by National Science Foundation grant PCM 78-13241.     

Female-Biased Attraction of Oriental Fruit Fly, Bactrocera dorsalis (Hendel), to a Blend of Host Fruit Volatiles From Terminalia catappa L.

Coupled gas chromatography-electroantennogram detection (GC-EAD) analysis of volatiles from tropical almond fruit, Terminalia catappa L., revealed 22 compounds that were detected by antennae of oriental fruit fly females, Bactrocera dorsalis (Hendel). Both solid-phase microextraction (SPME) and Porapak Q were used for sampling odors in fruit headspace, with SPME collections producing larger EAD responses from a greater number of compounds. Geranyl acetate and methyl eugenol elicited the largest EAD responses. A synthetic blend containing SPME collected, EAD stimulatory compounds showed female-biased attraction in laboratory wind tunnel bioassays, but heavily male-biased trap captures in a larger olfactometer arena. A nine-component subset of compounds eliciting relatively small EAD responses (EAD minor) and consisting of equal parts ethanol, ethyl acetate, ethyl hexanoate, hexyl acetate, linalyl acetate, ethyl nonanate, nonyl acetate, ethyl cinnamate, and (E)-β-farnesene, attracted mainly females. This EAD minor blend was as attractive to females and much less attractive to males when compared to torula yeast in field cage experiments using glass McPhail traps. Similar results were obtained with outdoor rotating olfactometer tests in which the EAD minor blend was almost completely inactive for males.