Floral Phenylpropanoid Cocktail and Architecture of Bulbophyllum vinaceum Orchid in Attracting Fruit Flies for Pollination
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- Tan, K.H., Tan, L.T. & Nishida, R. J Chem Ecol (2006) 32: 2429. doi:10.1007/s10886-006-9154-4
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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.
KeywordsBulbophyllum vinaceumOrchidaceaePhenylpropanoidsFruit flyBactrocera dorsalisB. unimaculaTephritidaeSynomoneSex pheromonePollinationDynamic lip mechanism
A number of orchid species in the genus of Bulbophyllum (Orchidaceae: subfamily Epidendroideae, subtribe Bulbophyllinae) exhibit an elaborate floral architecture, in addition to characteristic floral fragrances, that attract and bring specific pollinators to the right position for effective pollination. Some of the species, here called “fruit fly orchids,” selectively attract fruit flies of the genus Bactrocera (Diptera: Tephritidae) with specific floral volatiles that act as a synomone. It has been reported that the ginger orchid (Bu. patens King) flower releases a ginger essence—zingerone, as a floral synomone that attracts fruit flies sensitive to both methyl eugenol (ME) and raspberry ketone (RK)—inclusive of both pestiferous and nonpestiferous species (Tan and Nishida, 2000). In addition, flowers of Bu. apertum Schltr. (subspecies verrucatum) release RK that attracts RK-sensitive Bactrocera species (Tan and Nishida, 2005). Furthermore, the fruit fly orchid flower (Bu. cheiri Lindl.) possesses several phenylpropanoids, of which the major component is ME. It attracts male flies of Bactrocera papayae Drew and Handcock that assist in its pollination (Tan et al., 2002; Nishida et al., 2004). [It is noted that B. papayae is neither a distinct biological nor genetic species different from the Oriental fruit fly, B. dorsalis (Hendel) (Naeole and Haymer, 2003; Tan, 2003) and henceforth it is referred to as B. dorsalis.] The fruit fly orchid also attracts males of B. carambolae Drew and Hancock, B. umbrosa (Fabricius), and the hybrid of B. dorsalis × B. carambolae. None of the above species produces nectar; hence the question of the reward for attracted fruit flies remains unanswered. Flies consume and then either convert the chemical attractant into male sex pheromonal component(s)—in the case for ME-sensitive species, or directly sequester the attractant as part of a male pheromone system in the RK-sensitive species (Nishida et al., 1988, 2004; Tan, 2000; Tan and Nishida, 1995, 1998, 2000, 2005; Tan et al., 2002). Hence, for the three Bulbophyllum species—Bu. patens King, Bu. apertum, and Bu. cheiri Lindl.—the respective floral attractants are themselves the rewards for the attracted flies.
The majority of the Bulbophyllum species are epiphytes found in the virgin pan-tropical forest (Vermeulen, 1991) in lower montane forest at ca. 1000 m above sea level. The vinaceous orchid, Bu. vinaceum Ames & C. Schweinf., is a rare epiphytic plant, that is endemic to the highlands of Borneo Island, such as the Crocker Range and Mt. Kinabalu of Sabah, East Malaysia. Its name is derived from the Latin word “vinaceus” (for dark wine red) (Vermeulen, 1991) due to its deep wine-red colored, single-flowered inflorescence. Its resupinate flower (with lip turned downwards) has a protruding and stiff hamulus (pollinia stalk), whose function is unknown (Rasmussen, 1985), and a “spring loaded” hinged lip that is always held in a closed position (presumably protecting its sexual organs). The flower has a mild sweet scent resembling that of an ester and strongly attracts males of B. dorsalis.
The objectives of this paper are to investigate: (1) the behavior of fruit fly visitors and determine species identity of attracted flies; (2) the process of pollinarium removal as well as to determine the role of floral hamulus and the constantly closed lip; (3) the chemical constituents in floral fragrance that attract fruit flies; and (4) the content of phenylpropanoids in various floral organs and rectal gland of fruit fly visitors.
Methods and Materials
Observations of Flies on flowers
Observations of orchid flowers and plants were conducted in Kundasang (ca. 1800 m at the foothill of Mt. Kinabalu), Sabah, East Malaysia, and in Tanjung Bungah (lowland <200 m above sea level), Penang (plants originally obtained from Kundasang). Fly attraction was observed continuously from 07:00 to 17:00 hr on the day a flower bloomed. Investigations of pollinarium removal by a fruit fly were conducted when the first fly visited and fed on a newly opened flower; this process was videotaped whenever possible. Attention was paid to the role of the floral lip (labellum) and hamulus during pollinarium removal. To confirm that fruit flies were attracted to the flower via scent and not visually via floral color or form, certain investigations were performed with the fully developed bud covered with a piece of black mosquito wire netting; the flower was covered at dawn before it bloomed. After observation, flies on the flowers were collected in clear plastic bags, and their respective species identity was confirmed, if necessary, under 40× magnification. To ascertain floral response to lower light intensities especially during the night, newly bloomed flowers were left on the plant for further observation (after exposure to fruit flies). Observations were conducted on at least three flowers.
Volatile components were analyzed via gas chromatography-mass spectrometry (GC-MS) using an HP 5989B mass spectrometer coupled with an HP 5890 series II plus gas chromatograph equipped with an HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness) programmed from 60°C (1 min holding) to 280°C at a rate of 10°C/min. Quantification of volatiles was done on an HP 4890A gas chromatograph using an HP-1MS capillary column (20 m × 0.25 mm, 0.25 μm film thickness) and programmed from 80°C (1 min holding) to 280°C at a rate of 10°C/min; the GC was equipped with a total ion monitor and flame ionization detection (FID) using internal standards [1-tetradecanol or ethyl 4-(3,4-dimethoxyphenyl)-propanoate].
Volatile components from Bu. vinaceum flowers were collected during the day by using a glass jar (250 ml) with a filtered air inlet at the bottom end—connected to an aquarium pump (air flow regulated at ca. 20 ml/min)—and an outlet at the top end—directly connected to a TENAX column (packed with 100 mg in a glass tube). The TENAX column was replaced every 2 hr and washed with 0.5 ml ethanol. The resulting eluate was partially concentrated under reduced pressure (20 mmHg, <20°C) and then subjected to GC-MS and GC-FID analyses, with 1-tetradecanol as an internal standard.
Extraction of Floral Volatiles
Bu. vinaceum plants were originally collected in Kundasang, Sabah, and grown under outdoor conditions in a home garden in Tanjong Bungah, Penang, Malaysia, to obtain the blossoms. Flowers were plucked within 4 hr of blooming (07:00–9:00 hr), weighed, and immersed in sufficient redistilled ethanol in a 20-ml glass vial, and then used for GC quantifications. For individual floral organs (petals, lateral and medial sepals, lip, and column), each part was carefully removed from a freshly bloomed flower (within 3 hr after it bloomed and not exposed to fruit flies), weighed, and soaked in sufficient ethanol in a 5-ml glass vial for quantification of ME.
Extraction of Volatiles from Fruit Flies
Ten wild flies, which were observed feeding on the floral tissues of Bu. vinaceum flower, were captured in the orchid habitat in Kundasang, Sabah (October 1, 2002). Flies (captured in the afternoon) were kept alive until the evening (ca. 6 hr—sufficient time to incorporate and sequester the phenylpropanoids into the rectal pheromone glands)—and then dissected. The rectal gland from each fly was removed and individually soaked in ethanol (0.25 ml in 1-ml vial). Each fly body received the same treatment. A laboratory-reared male (18 d old, and not previously exposed to any phenylpropanoids) of B. dorsalis was processed in a similar manner after feeding 10–20 min on a flower of a potted Bu. vinaceum plant in Penang (October 2003) and kept alive for 24 hr before rectal gland extraction.
Eugenol and methyl eugenol were purchased from Tokyo Chemical Industries (Osaka, Japan). 2-Allyl-4,5-dimethoxyphenol, euasarone (5-allyl-1,2,4-trimethoxybenzene), cis- and trans-coniferyl alcohol, and trans-3,4-dimethoxycinnamyl alcohol were synthesized as previously described (Tan and Nishida, 1998; Nishida et al., 2004).
Fruit Fly Species
In the highlands of Kundasang, males of two sibling species belonging to the B. dorsalis complex—B. dorsalis (most abundant) and B. unimacula Drew and Hancock visited flowers of Bu. vinaceum. Many males were observed feeding on a single flower and at times, the flower was completely covered with attracted flies (>20). The number of visitors dwindled towards dusk. Of the 10 flies captured from a single flower for rectal chemical analysis, eight were B. dorsalis and two were B. unimacula; all were male.
A similar phenomenon was observed in the lowland (nonendemic area), but with one or two individuals of B. umbrosa and B. carambolae occasionally joining the abundant B. dorsalis and the wild hybrid (between B. dorsalis and B. carambolae; Wee and Tan, 2005). Besides being major agricultural pests, all three are ME-sensitive species that are known to respond to ME and have been captured in ME baited traps (Tan and Lee, 1982). No other organism or insect visited the flower. When a flower was covered with fine black wire netting—through which neither the shape nor the color of the flower can be distinguished—flies landed on the netting and were observed to frantically search before settling on the area of netting nearest to the flower. This indicated that they were attracted by the fragrance and not the floral color or shape. No female flies were attracted to the flower, irrespective of time or location.
Apparently shocked and shaken, the fly retreated from its temporary confinement between the closed lip and the floral column—by backing out towards the tip of the lip, hence pivoting the lip open again. Figure 1e and f shows the fly exiting the flower with the hamulus and the pollinia (ca. 2 mm from the surface of the fly’s thorax) stuck to it. The whole process of actual pollinarium dislodgement from the anther and its adhesion to the fly was precise, smooth, and nearly instantaneous—occurring in less than 1 sec (as determined from video footage). Once free, the fly either took off (one in five observations) or dismounted from the lip and continued to settle on the flower (Fig. 1f) while intermittently feeding, resting, preening, and cleaning its proboscis for 6–40 min if left undisturbed—until satiation. In the latter situation, no self-pollination was observed.
As the fly dismounted or took off from the opened lip, the lip immediately sprang back to its normal closed position (in less than 0.02 sec—the speed of a single frame of the video clip). In all five cases observed, it was the first fly that landed on the flower that removed the pollinarium; and because there is only one pollinarium, subsequent flies regardless of number were just visitors (>35 fruit flies per flower/d) feeding on floral fragrance on all parts of the flower.
The flower partially closed at night. This process took several hours beginning in late afternoon nearing sunset. The partially closed flower reopened with its petals and sepals spread out again the next day (10:00–12:00 hr).
Identification of Phenylpropanoids
Compound 1 (eugenol) GC: Rt (min) 11.10; m/z(%) 164(100, M+), 149(35), 137(17), 131(24), 121(14), 104(14), 103(22), 91(16), 77(19).
Compound 2 (methyl eugenol) GC: Rt (min) 11.67; MS: m/z(%) 178(100, M+), 163(33), 147(18), 135(10), 107(21), 103(20), 91(21), 77(9).
Compound 3 (euasarone). GC: Rt (min) 13.77; MS: m/z(%) 208(100, M+), 193(53), 177(20), 165 (14), 133(10), 124(10), 105(7), 91(9), 77(7).
Compound 4 (2-allyl-4,5-dimethoxyphenol). GC: Rt (min) 14.33; MS: m/z(%) 194(100, M+), 179(87), 163(9), 151(12), 123(31), 91(17), 77(12), 69(12).
Compound 5 (cis-coniferyl alcohol). GC: Rt (min) 14.93; MS: m/z(%) 180(70, M+), 152(15), 147(10), 137(100), 124(42), 119(23), 103(15), 91(32), 77(19).
Compound 6 (trans-coniferyl alcohol). GC: Rt (min) 15.67; MS: m/z(%) 180(82, M+), 152(10), 147(10), 137(100), 124(48), 119(24), 103(12), 91(31), 77(14).
Compound 7 (trans-3,4-dimethoxycinnamyl alcohol). GC: Rt (min) 16.08; MS: m/z(%) 194(97, M+), 177(14), 165(17), 161(23), 152(19), 151(100), 138(55), 119(16), 107(12), 91(33), 77(28).
Compound 8 (trans-3,4-dimethoxycinnamyl acetate). GC: Rt (min) 17.40; MS: m/z(%) 236(100, M+), 193(41), 177(59), 165(31), 146(59), 138(18), 119(10), 105(10), 103(10), 91(16), 77(9), 43(26).
Emanation of methyl eugenol (2) was confirmed by GC-MS analysis of the headspace of a whole flower of Bu. vinaceum (diagnostic ion mass chromatogram: m/z 178, 163, 147, 107, 103, and 91; Rt=11.67 min). Although the exact emission rate could not be determined due to low recoveries, methyl eugenol emanation from a flower (1 and 2 d old) was roughly estimated in a range of 10–100 ng/2 hr. No other phenylpropanoid analogs contained in the flower tissues were detected.
Distribution of Phenylpropanoids in Floral Organs
Accumulation of Floral Volatiles in Rectal Glands
A laboratory-reared virgin male of B. dorsalis, which received the pollinarium on his thorax after voracious feeding on Bu. vinaceum flower, sequestered 2.3 and 3.0 μg of compounds 4 and 6, respectively, in the rectal glands.
Of the 10 males captured on a Bu. vinaceum flower under natural conditions in Sabah, eight flies were identified as B. dorsalis and two as B. unimacula. Among the B. dorsalis males, six of them possessed relatively low quantities of compound 4 (0.51±0.18 μg/male) and a trace amount of 6 (less than 0.10 μg/male), whereas the other two possessed 24.5 and 45.7 μg of compound 4, and 0.5 and 4.9 μg of compound 6, respectively. Both B. unimacula males captured on the same flower possessed a trace amount (approx. 0.03 μg) of 4, and 0.99 and 0.49 μg of 6, respectively, in addition to large quantities of two sesquiterpenic compounds tentatively identified as β-caryophyllene and humulene. These flies appeared to have obtained large quantities of the sesquiterpenes from other plant sources prior to visiting Bu. vinaceum.
Both Bu. vinaceum and Bu. apertum flowers have architectures that include prominently protruding hamuli and hinged floral lips. According to Rasmussen (1985), the function of the prominent hamulus for Bu. ecornutum (=Bu. apertum) is unknown. However, observations of Bu. vinaceum presented here demonstrate the function of the hamulus as a “crowbar” to pry the pollinia out of its protective anther cover.
The morphology of the floral lip in Bu. apertum—except for the fact that it is hinged—is like any other petal, i.e., in the open position. Hence, the reproductive organs are always exposed and can be easily or accidentally removed by any part of the fly. Furthermore, the pollinarium once removed can just as easily be deposited anywhere on the petals or medial sepals of the same flower, leading to a total wastage of pollen (Tan and Nishida, 2005). Hence, the function of the “spring loaded” ovately shaped floral lip of Bu. vinaceum—always in the closed position—presumably is to protect the pollinarium from accidental removal.
A weight comparison of the portions of the Bu. vinaceum lip on either side of the hinge/pivot point demonstrated the presence of a nominal spring restoring force that retains the lip in its normally closed position (Tan, unpublished data). Based on two different methods of calculations performed on preserved flowers, the rotational force required to catapult the fly to the observed velocities was estimated to be between 117 and 122×10−9 N m (Tan, unpublished data). Additionally, the fact that the highest concentration of chemical reward occurs on the adaxial side of the lip ensures that an attracted fly will eventually end up here.
Of all the other Bulbophyllum species studied so far, this catapulting lip and stiff crowbar-like hamulus is unique to Bu. vinaceum. In contrast, other Bulbophyllum species, which possess flexible stipes instead of hamuli, such as Bu. macranthum, Bu. cheiri, and Bu. patens, have floral lips that act like seesaws and are normally in an open position. In Bu. macranthum, the slippery inner edges of the lateral sepals cause the fly to fall onto the lip, which then tips into the closed position sending the fly—abdomen first—into the column cavity (Ridley, 1890). For Bu. cheiri and Bu. patens, the floral lip is also usually in an opened position seesawing gently with the breeze. The fly settles on the lip to feed on chemical attractant, and as it moves past the point of imbalance the lip tilts suddenly to a closed position. Thus, for these two species, the fly is sent headfirst into the column cavity (Tan and Nishida, 2000; Tan et al., 2002). The only other species investigated with a hamulus is Bu. apertum (Tan and Nishida, 2005). However, in this flower the hamulus is exposed because of the absence of a spring loaded lip; instead, it has a small triangular seesaw lip that is not large enough to protect the flower’s sexual organs.
In Bu. vinaceum, the speed of pollinarium detachment and precision of attachment to the fly’s dorsum is remarkable. This dynamic process is likely assisted by the stiff hamulus, which allows the momentum of the catapulted fly to be converted into a force that pries out the pollinia from the anther. Conversely, pollinarium removal in Bu. baileyi is a much slower process, taking at least 4 orders of magnitude longer (up to 46 min). In this process, the fly adheres to the stipes of the pollinarium, which is attached to the anther, and is hence suspended from the pollinarium. It has to struggle vigorously with wing and leg movements, occasionally clinging onto the small seesaw lip, in order to free itself, and thus, in the process, loosens the pollinia from the anther (Tan, unpublished observation). The adhesion of the Bu. baileyi pollinarium to the fly is so strong, that it is believed to be permanent, as reported by Smythe (1969), where a fly—after depositing the pollinia onto the stigma of another flower—remained attached to the pollinarium and suspended from the stigmatic surface until death.
Methyl eugenol (2) and trans-coniferyl alcohol (6) are the two volatile components with the highest concentrations in Bu. vinaceum flowers, accompanied by a series of related phenylpropanoids (1, 3, 4, 5, 7, and 8). However, in another methyl eugenol-producing species, the fruit fly orchid, Bu. cheiri, the floral content of 2 varies from 85% to 90% of the total phenylpropanoid volatiles, with some minor ingredients similar to those found in Bu. vinaceum (Tan et al., 2002; Nishida et al., 2004). While 2 is known as a potent male attractant for B. dorsalis, compounds 4 and 6 and their derivatives 7 and 8 are moderate attractants for male flies and induce compulsive feeding behavior (Tan and Nishida, 1998; Khoo et al., 2000). Compounds 4 and 6 quickly build up in the rectal glands, when the male flies ingest 2 (Nishida et al., 1988). In contrast, if males are fed artificially either with compounds 4 or 6, these compounds are sequestered unchanged in the rectal glands (Nishida et al., 2004). Compounds 7 and 8 are hydrolyzed by the flies to 6 after ingestion (Nishida et al., 1997). Both rectal compounds 4 and 6 serve as a sex pheromone and attract females during the courtship period at dusk (Tan and Nishida, 1998; Khoo et al., 2000). Thus, the phenylpropanoid cocktail in the Bu. vinaceum flower provides both intact pheromone materials (4 and 6) and their precursors (2, 7, and 8) to the male flies. According to the headspace analysis, methyl eugenol (2) was the major volatile. The emission rate is sufficient to attract male Bactrocera flies to the flower as the flies are extremely sensitive to this compound (ca. 1 ng spotted on a TLC plate was able to attract one or two native B. dorsalis males in the field; Tan, unpublished data). It is likely that the flower emits only a small amount of 2 that acts as a long distance signal, and then, provides a variety of less volatile compounds (e.g., 4, 6, 7, and 8) in its floral tissues that guide the attracted fly towards the lip area where the concentration of phenylpropanoid ingredients is the highest. This demonstrates that the floral volatiles act as synomone in this interaction in which both flies and orchid flowers gain reproductive benefits, i.e., the flower’s pollinarium is transported for pollination, and the male fruit fly is rewarded with the chemicals that are used for the production of its sex pheromone (Tan and Nishida, 2000; Tan et al., 2002).
The behavioral and physiological activities (or functions) of phenylpropanoids (1–8) found in Bu. vinaceum flowers—evaluated from previous work on B. dorsalis (Nishida et al., 1988, 1997; Tan and Nishida, 1998; Khoo et al., 2000) are as follows: compounds highly attractive to males: 2; compounds moderately attractive to males: 4, 6, 7, and 8; compounds marginally attractive to males: 1, and 3; compounds with potent phagostimulant activity toward males: 2, 4, 6, 7, and 8 (5 not tested); compounds sequestered into the male rectal organ after ingestion of methyl eugenol (2): 4 and 6; compounds biotransformed to coniferyl alcohol (6) in the crop after ingestion: 2, 7, and 8; compounds attractive to females during courtship period as a sex pheromone: 4 and 6.
The reason why Bu. vinaceum flowers produce such an assortment of phenylpropanoids—in addition to the most attractive compound (2)—is puzzling. The less volatile phenylpropanoids may function as short-range attractants (olfactory) or phagostimulants (gustatory) to arrest male flies, at the same time securing the endowment of the pheromone precursors to the faithful pollinators. Although the flower produces components 4 and 6, which attract and arrest females during courtship and hence would serve as specific “female attractants,” the flower has never been observed to attract female fruit flies—not even during dusk when females are the most sensitive to these chemicals. This absence of attracted females despite the presence of female attractants is an unusual phenomenon—perhaps, it is because floral emission rate of compounds 4 and 6 declines through the day. Furthermore, it is possible that some of the compounds are simply by-products of other processes and, as such, do not have an explicit (or evolved) function in fly attraction. Hence, at this point, we do not have a conclusive answer for the function(s) of all the compounds produced by Bu. vinaceum. However, we believe that the original mutualistic interactions between Bu. vinaceum and its “true” partner (probably B. dorsalis) could shed some light. The hypothesis that the flower and its true partner coevolved, thus allowing the flower to blend the fly’s favorite cocktail in order to charm the male fruit flies is currently being investigated.
We express our thanks to Jaap J. Vermeulen of National Herbarium Netherlands, Leiden Branch for useful information on Bu. vinaceum. This work was partially supported by the Grant-in-Aid for Scientific Research from JSPS (No. 15405022) and a Grant-in-Aid for the 21st Century COE Program for Innovative Food and Environmental Studies Pioneered by Entomomimetic Sciences, from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We also thank Diogo Ezequiel and Kayin Dawoodi for their work on dynamic flower mechanisms.