ATTRACTION OF FEMALE AND MALE Bactrocerapapayae TO CONSPECIFIC MALES FED WITH METHYLEUGENOL AND ATTRACTION OF FEMALES TO MALE
SEX PHEROMONE COMPONENTS
ALVIN KAH-WEI HEE* and KENG-HONG TAN
School of Biological SciencesUniversiti Sains Malaysia11800 Penang, Malaysia
(Received August 13, 1997; accepted December 15, 1997)
Abstract—The attraction of female and male Bactrocera papayae to conspe-cific males fed with methyl eugenol (ME) and female attraction to male syn-thetic sex pheromone, fra/w-coniferyl alcohol (CF), were evaluated in a windtunnel. Earlier and greater attraction were exhibited by both females and malesto ME-fed than to non-ME-fed males as dusk approaches. Males increasedtheir precopulatory behavior (i.e., wing fanning and mounting) during theperiod of higher attractancy. These data confirm that the consumption of MEenhances the mating competitiveness of males and suggest that ME also func-tions as a precursor to the male sex and aggregation pheromones. Threephenylpropanoid compounds biosynthesized from ME, coniferyl alcohol,2-allyl-4,5-dimethoxyphenol, and 3,4-dimethoxycinnamyl alcohol, weredetected in male rectal gland along with an endogenous rectal compound,/V-(3-methylbutyl) acetamide. When offered singly to the females, coniferylalcohol was found to be most attractive.
B. dorsalis Hendel complex. In this complex, several species are regarded asmajor or potentially important pests that pose a significant threat to the agricul-tural industry in subtropical and tropical countries. In Malaysia, B. papayas isa serious pest infesting many commercially grown fruits (Tan and Lee, 1982).
In order to control these fruit fly species, methyl eugenol (ME), a highlypotent male attractant, was extensively used with great success especially inmale annihilation programs (Steiner et al., 1965). Recently, it was found thatthe consumption of ME enhances the mating competitiveness of males (Shellyand Dewire, 1994; Tan and Nishida, 1996).
Chemical analyses showed that B. papayae males converted ME to 2-allyl-4,5-dimethoxyphenol (allyl-DMP), which accumulated in larger amounts, andto relatively smaller amounts of cis- and frans-coniferyl alcohol and c/s-3,4-dimethoxycinnamyl alcohol (cw-DMC). These compounds were found in therectal gland (Nishida et al., 1988a,b; Tan and Nishida, 1996). An amide com-pound, AK3-methylbutyl) acetamide (W-MBA), was detected in trace amountsand reported as a minute volatile component of the rectal gland (Perkins et al.,1990). frans-Coniferyl alcohol (CF) has been identified as a male sex pheromone(Tan and Nishida, 1996). It was found to attract conspecific females at closerange and stimulated ovipositor extrusion at the point of highest concentration.Similarly, at close range males were also strongly attracted to and feed on thecompound (Tan and Nishida, 1996). The rectal compounds were released intothe air during dusk, which coincided with the period of fruit fly courtship (Nish-ida etal., 1988a).
In light of the possibility of using sex pheromones as a tool for monitoring,surveillance, and control of female fruit flies, information on the attractancy ofthe male-produced sex pheromone in ME-fed B. papayae is still insufficient.Here, we report on the attraction of female and male B. papayae to conspecificmales fed with ME, and female attraction to male synthetic sex pheromone andrectal gland compounds.
METHODS AND MATERIALS
Insects. Laboratory-reared B. papayae larvae were cultured on an artificialmedium containing yeast, sugar, bran, sweet potato, and water. Adult flies wereprovided with a mixture of water and yeast, protein hydrolysate, and sugar.Males and females were segregated by sex one to three days after emergence(DAE). The flies were maintained under ambient conditions in the insectary ona 12L: 12D cycle at 25-29°C and 83-90% relative humidity. Sexually maturevirgin males (14-20 DAE) that responded maximally to ME (Tan et al., 1987)and 14-20 DAE females were used for experimentation.
Chemicals. CF and compounds allyl-DMP, m-DMC and Af-MBA used forchemical attractancy tests were provided by R. Nishida, Kyoto University, Japan.
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All the compounds used were pure compounds except trans-CF and cw-DMC,which were 96.0% trans and 97.0% cis, respectively. The compounds used forthe bioassays were dissolved in absolute ethanol to a concentration of10 mg/ml.
Live-Fly Pheromone-Source Test. Laboratory bioassays were conducted ina wind tunnel previously described by Jones et al. (1981) and modified by Tanet al. (1987). It is constructed of a transparent polythene tube (0.55 m diam. X4 m) and has two end grills spaced 2 m apart. Air speed was regulated at 12-15cm/sec by voltage regulators connected to fans. Fumes of ammonium chloridewere used to measure air speed. To prevent possible contamination, the plastictubing was changed regularly and the grills, wire meshes, and fan blades werethoroughly cleaned with ethanol. Experiments were conducted in a researchlaboratory that received natural daylight from the south. The temperature in thelaboratory was 27-30 °C and relative humidity 83-91%. The wind tunnel wasplaced on a workbench beside the windows.
In the live-fly attractancy tests, 10 sexually mature virgin males were usedas a pheromone source. The flies were fed with ME from 08:30 to 09:00 hrduring their peak period of response to ME (Tan, 1985) before they were usedas test subjects in a wind tunnel in late afternoon and dusk (16:45-19:40 hr).Each male was given 0.5 n\ of ME on a Whatman No. 1 filter paper placed ona Petri dish (9 cm diam.), in a laboratory situated ca. 50 m from the insectary.Only males that responded and fed on ME were selected. After consuming ME,the flies were returned to a cylindrical stainless steel cage that had both endscovered with plastic Petri dishes.
Prior to experimentation, sexually mature virgin flies to be used for testingor as a pheromone source (live-fly attractancy tests) were transferred to cylin-drical plastic (9 cm diam. x 12 cm) and stainless steel cages (9 cm diam. x12 cm). Each cage contained food and water. The cages were placed in theresearch laboratory overnight for the flies to acclimatize to test conditions. Foodand water were removed from the cages 30 min before the experiments, andcaged ME-fed males were placed in the upwind area of the wind tunnel foracclimatization. The cage was laterally aligned to the upwind grill and wasplaced on a tripod 20 cm high and 20 cm from the upwind end of the windtunnel.
In the female attraction studies, 15 sexually mature females were releasedat the downwind end of the wind tunnel from a cylindrical plastic cage withgreen mesh ( 8 x 5 cm) positioned perpendicular to the downwind grill. Thecage was supported at 20 cm height by a tripod and situated 1.7 m from thesource of attraction. A positive response was recorded when a zigzag flight[zigzagging anemotaxis as defined by Kennedy and Marsh (1974)] over at least50 cm was performed by a fly to an active upwind section of the tunnel (40 cmfrom the upwind grill). In the first trial, the females were released at 16:45 hr,
755B. papayae ATTRACT ANTS
and responses were recorded after 10 min. Subsequently, an interval of 5 minwas allowed to collect the released flies. Clean specimen vials (1.0 cm diam.X 5.5 cm) were used to collect the released flies. The procedure was repeatedwith different flies to avoid habituation. Trials were conducted from 16:45 to19:30 hr. The average corresponding light intensities in the upwind and down-wind area were measured with a lightmeter, ANA-999 (Tokyo Photo-ElectricCo. Ltd., Japan). The experiment was replicated three times on different days.Non-ME-fed males were used as a pheromone source in control experiment.The control experiment was conducted separately from the treatment experimenton different days. Male attraction studies were conducted by a procedure thatwas similar to the procedure described for females, except that caged maleswere used to assay for their attractancy to sexually mature males instead offemales.
Chemical Attractancy Test. Laboratory bioassays of the attraction of sex-ually mature females to individual compounds of CF, allyl-DMP, cis-DMC,and N-MBA were conducted before sunset, from 18:15 to 19:15 hr. For eachof the bioassays, 3 ul of the solution (concentration = 10 mg/ml in absoluteethanol) were dispensed from a Hamilton 25-ul syringe on a Whatman No. 1filter paper (4.25 cm diam.) placed on an inverted glass Petri dish that wassupported by a tripod 20 cm high and 10 cm from the upwind end of the windtunnel. Five microliters of pure distilled water were then dispensed on the com-pound to enhance the evaporation rate. Fifteen sexually mature females werereleased into the wind tunnel. The chemical tests were conducted in a similarmanner to the live-fly pheromone-source tests except that the sample solutionswere used as test subjects and after each bioassay the filter paper that containedthe chemical was discarded and the glass petri dish thoroughly cleaned withethanol to prevent contamination. As controls, the tests were repeated withabsolute ethanol in place of the sample solutions. During experimentations,lighting was maintained at ca. 34 lux by fluorescent lights to facilitate mea-surement and dispensing of the sample solutions. Six replications of the exper-iment were performed.
Percentages were transformed to a modified arc-sine square root and werethen subjected to analysis of variance. Means were separated by Student's t testand Duncan's multiple range test.
RESULTS
Live-Fly Pheromone-Source Test. Our results show that ME-fed males usedas the pheromone source attracted more conspecific flies of both sexes thanmales not fed with ME (P < 0.01) (Figures 1 and 2). When females weretested against conspecific ME-fed males, the peak attractancy was observed to
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B. papayae ATTRACTANTS
FIG. 1. Mean response of female Bactrocera papayae to caged conspecific males in awind tunnel; four replicates. Vertical bars represent standard errors; comparison of meanswith Student's t test (P = 0.01).
FIG. 2. Mean response of male Bactrocera papayae to caged conspecific males in awind tunnel; four replicates. Vertical bars represent standard errors; comparison of meanswith Student's t test (P = 0.01).
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occur earlier (at 18:55 hr; ca. 40 min before sunset), under average light intensityof 117 lux compared with responses of females to control males, which peakedat 19:10 hr under light intensity of 55 lux (Figure 1). Maximal responses offemales to ME-fed and control males decreased after a drop of ca. 40-60 luxin light intensity. When light intensity was less than ca. 10 lux, females did notexhibit higher percentages of response to ME-fed males (0.20 < P < 0.25).Similarly, when responses of males to conspecific males were assayed (Figure2), ME-fed males attracted higher percentages of males, with a peak occurringat approximately 18:55 hr under an average light intensity of 94 lux, in com-parison to the attractancy of males to control males, which peaked at 19:10 hrat a light intensity of 51 lux. Subsequently, a drop of ca. 40 lux correspondedto a decrease in the responses of males to conspecfic ME-fed and control males.As the light intensity decreased to less than 10 lux, males also showed nosignificant difference (0.30 < P < 0.40) between the attractancy to conspecificME-fed and control males (Figure 2). ME-fed males were equally attractive tomales and females (Figure 1 and 2). Similar results were also observed whenmales and females were compared for responses to conspecific control males.
Observations of the caged ME-fed and control males used as pheromonesources revealed that peak attraction responses of males and females corre-sponded with the calling activity in the cages. Approximately 50% of the cagedmales moved and flew rapidly in an erratic manner. This increase in the loco-motor behavior was also accompanied by an increase in physical contact, wheremales would often engage in head-on collisions and "boxing" behavior. Suchdisplays were observed when males exhibited aggressiveness in defending theirindividual territories. Males engaged in wing fanning and attempts to mount andcopulate with other males. Two types of wing fanning were observed: one typewas just prior to mounting accompanied by the rhythmic beating of the abdomenby the rear legs; and the second type occurred as the male settled onto the backof the other male in mounting attempts. Released flies that responded to thesexually excited males would orient to the source and perform zigzagging ane-motaxis. Upon locating the source, the flies would either land on the cage, thesides of the polythene tube, or the mesh of the upwind grill. We also observedthat some females extruded their ovipositors after landing on the cage containingME-fed males. However, no copulations between the caged control males andreleased conspecific females occurred.
In the male attractancy to conspecific males, released males were alsoobserved to fan their wings and a small number (ca. 10%) attempted to mounteach other. When light intensity decreased to less than 50 lux, the mating activityof the caged ME-fed and control males declined rapidly. The caged malesremained passive and displayed only occasional locomotion behavior.
Chemical Attractancy Tests. The response of virgin females of CF shows
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B. papayae ATTRACTANTS
FIG. 3. Mean response of female Bactrocera papayae to frans-coniferyl alcohol (CF)placed at different distances in a wind tunnel; six replicates: Vertical bars representstandard errors, bars having the same letter are not significantly different at P = 0.01;comparison of means by Duncan's multiple-range test.
that over 40% responded by zigzag anemotaxis to the source when released froma distance of less than 1.3 m (Figure 3). When CF was placed at distancesbetween 0.5 and 1.1 m, it attracted significantly higher percentages of femalescompared with distances more than 1.5 m (P < 0.01) (Figure 3). Femaleresponses to individual compounds of the male rectal glands are compared inFigure 4. ME-fed and control males (at peak response times) attracted signifi-cantly more conspecific females than were attracted to individual compounds ofCF, allyl-DMP, cis-DMC, and W-MBA (P < 0.01) (data obtained from Figure1). Attraction of virgin females to CF, allyl-DMP, cw-DMC, and N-MBA waslow, with less than 20% of flies responding by zigzagging anemotaxis. Of allthe individual compounds tested, CF was shown to be most attractive to thefemales.
In all the chemical attractancy tests, only a small number (ca. 1%) offemales actually landed on the source. However, sexually receptive femaleswould respond by zigzagging anemotaxis to the active upwind section. The fliesappeared to have flown against the wind to the active section by locating thepheromone plume. Upon missing the plume, the flies would normally land onthe sides of the polythene tubing.
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FIG. 4. Mean response of female Bactrocera papayae to conspecific males and variouschemicals placed 1.7 m from the flies in the wind tunnel; six replicates. Data on ME-fed and control males, respectively, obtained from Figure 1. Absolute ethanol was usedas control. Vertical bars represent standard errors, bars having the same letter are notsignificantly different at P = 0.01; comparison of means by Duncan's multiple-rangetest.
DISCUSSION
ME is a precursor to male sex and aggregation pheromones in B. papayae(Tan and Nishida, 1996). Similar results were also obtained through chemicalanalyses and behavioral studies on the Oriental fruit fly, B. dor salts, and anotherspecies in the B. dorsalis complex, B. carambolae (Tan and Nishida, 1996).Phenylpropanoids, CF, allyl-DMP, and cw-DMC, are male attractants toB. papayae and B. dorsalis (Nishida et al., 1988a; Tan and Nishida, 1996; Tan,1996). Allyl-DMP was most attractive, and cis-DMP least attractive when pre-sented to the males (Tan, 1996). The phenylpropanoids are accumulated in themale rectal gland (Nishida et al., 1988a). Compounds CF and allyl-DMP wereimmediately detected in the rectal gland after males consumed ME. In our study,the phenylpropanoids were released 10 hr after the males consumed ME, whichcorresponded to the period when ME-fed males engaged in wing-fanning andmounting. Our results suggest that B. papayae males release the pheromone bywing fanning before and during mounting attempts to copulate. Decreasing lightintensity as dusk approaches is regarded as the normal stimulus required for theflies to initiate mating behavior. Observations of ME-fed males in a field cage
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(2.1 X 2.1 x 2.1 m) showed that virgin females were attracted to and fed onthe rectal gland secretion of males in copula with other females (unpublisheddata).
The mating behavior of caged males used as a pheromone source resemblesthat of B. dorsalis (Kobayashi et al., 1978). B. papayae males utilize a lekmating system, a communal display area where males congregate to attract andcourt females for the sole purpose of copulation (Emlen and Oring, 1977). Lekformation in the B. dorsalis complex has been proposed as a form of distractionby the male when releasing the sex pheromone, which simultaneously functionsas an aggregation pheromone, thereby diverting the attention of other malesfrom a potential female mate (Tan and Nishida, 1996).
Besides acting as a pheromone precursor, ME also confers a significantadvantage to males by functioning as an allomone precursor that deters predationby vertebrates. Observations have shown that when sexually mature B. papayaewere put under stress, the flies ejaculate rectal contents (Tan and Nishida, 1996).A deterrent effect was produced by ME-fed B. papayae males on the houselizard, Hemidactylus frenatus. In a no-choice experiment, after initial exposureto ME-fed males, the lizards prefer to starve to death rather than feed on theflies (Tan and Nishida, 1992). Studies on the Japanese tree sparrow, Passermontunus saturatus, revealed the increasing order of compounds CF < ME <allyl-DMP as feeding deterrent (Nishida and Fukami, 1990). In a choice feedingexperiment involving European starlings, Sturnus vulgaris, CF was the mostpotent repellent followed by 3,4-dimethoxycinnamyl benzoate among a groupof phenylpropanoids tested (Jakubas et al., 1992). Therefore, in B. papayae, itis probable that males also possess an antipredation mechanism through theendogenous production and secretion of an allomone.
There is no previously published report on tephritid male attractancy tomale-produced pheromone in a wind tunnel, although male-to-male attractionin the field is known for a number of species. This study has, for the first time,successfully demonstrated the strong response of B. papayae males to phero-mone from conspecific ME-fed males in laboratory bioassays in a wind tunnel.In the past, the possible reason for the lack of success at proving responses ofmales to pheromone in the laboratory was attributed to males themselves actingas treatments because they produce pheromone in the bioassay chamber. Theseunwanted pheromone sources may then compete with the experimental treat-ments and also convert controls to treatments (Robacker and Hart, 1986). Inthis wind tunnel study, the pheromone from males is allowed to flow againstreleased flies at a constant rate, thus ensuring that pheromone from the releasedmales does not act as treatment. Clean air that is used to carry the pheromoneis delivered from the inlet fan and is drawn out of the tunnel to the outsideenvironment. This further ensures that air delivered into the tunnel is not con-taminated.
761B. papayae ATTRACTANTS
Consumption of ME enhances the mating competitiveness of males. Thisis suggested by the strong attraction of females to conspecific ME-fed males inthe wind tunnel. In male-male mating competition for virgin females, malesthat fed on ME performed significantly better (Shelly and Dewire, 1994; Tanand Nishida, 1996). Preliminary results have also shown that ME-fed malesachieved earlier peak frequencies in wing fanning and mounting, and more than50% achieved copulation (unpublished data). Our work showed that femaleswere attracted earlier during dusk to ME-fed males. This probably accounts forthe higher mating success in ME-fed males compared with control males of thesame cohorts (Tan and Nishida, 1996). Thus, it appears that ME-fed malesproduced signals that were more attractive. CF alone failed to elicit significantlyhigher responses of females compared with live ME-fed males. This may becaused by the lack of other minor components.
The ME-fed B. papayae male has a multicomponent pheromone systemthat involves a combination of phenylpropanoids and other rectal compounds.Among the individual compounds tested, CF was the most attractive to virginfemales. Our work also showed that for female B. papayae in a confined space,CF elicited a significant percentage of zigzag anemotactic flight. When a mixtureof phenylpropanoid compounds CF and allyl-DMP was presented to female flies,it elicited higher percentages of response (Nishida, 1996). Therefore, to increasethe attractancy of CF, it needs to be blended with allyl-DMP, cw-DMC andW-MBA in the correct proportions. This requires further investigations.
In the chemical attractancy tests, conspecific females were also attracted to/V-MBA. In female B. dorsalis, N-MEA elicited zigzag anemotatic flight (Met-calf, 1990). N-MBA. was detected in rectal glands of a number of Bactroceraspp. (Fletcher and Kitching, 1995). Therefore, it appears that this endogenousrectal amide probably functions as a natural attractant or sex pheromone com-ponent that is enhanced by the presence of phenylpropanoids after pharmaco-phagy of ME.
The fact that conspecific females and males are sexually attracted to ME-fed males suggests the possibility of developing pheromone-based traps attract-ing both sexes in efforts to improve existing control strategies. Controlled-releaseformulations of the pheromone components are crucial to ensure their effective-ness in the field. Further work to determine the correct ratios of the pheromoneblend is currently being conducted.
Acknowledgments—We wish to thank the School of Biological Sciences, Universiti SainsMalaysia for providing the working facilities. We thank Dr. R. Nishida (Kyoto University) forproviding us with the chemicals. This work is supported by the Malaysian Government R & DGrant MPKSN/IRPA 01-02-05-6004.
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