Journal of Chemical Ecology. Vol. 22. No. 10. 1996

AGGREGATION PHEROMONES OF immigrans, D. phalerata, AND D. subobscura

KATARINA HEDLUND, 1.3. * ROBERT J. BARTELT,2 MARCEL DICKE,' and LOUISE E. M. VET'

'Department ofEll1omology. Wageningen Agricullllral University P.O. Box 8031.6700 EH Wageningen TIle Netherlands 2USDA-ARS National Cell1er for Agricultural Utilization Research. Bioactive COll.5!illle1l1S Research 1815 North University Street. Peoria. lllinois 61604 3Presell1 address: Departmell1 ofEcology. Lund Universit.v Ecology Building. 5-223 62 Lund. Sweden

(Received November 13. 1995; accepted May 16. 1996)

Abstract-Aggregation pheromones of Drosophila immigrallS. D. phalerata and D. subobscura were demonstrated by testing attraction of adult to hexane extracts of the flies in a windtunnel bioassay. Extracts of adult males of all species attracted conspecific males and females. However. D. subob­ scura flies were attracted only when the extract was applied on food. GC-MS analysis identified (Z)-Il-octadecenyl acetate (cVA) in the extracts of adult male D. immigrans and D. phalerata. Both species were attracted to synthetic cVA. Male and female D. subobscura produced 5.9-pentacosadiene. 5-pen­ tacosene. 2-methylhexacosene and 5.9-heptacosadiene. while only male D. subobscura produced (Z)-5-tricosene and minor amounts of cVA.

Key Words-Aggregation pheromones. (Z)-II-octadecenyl acetate. (Z)-5­ tricosene. 5.9-pentacosadiene. windtunnel. .

INTRODUCTION

Drosophila flies that breed in ephemeral habitats such as decomposing fruits and mushrooms often have aggregated distributions (Rosewell et al., 1990;

*To whom correspondence should be addressed.

1835

0098-0331/96/ I000·1835$09.50/0 1996 Plenum Publishing CorporJ.lion 1836 HEDLUND ET AL.

Jaenike and James, 1991). Aggregative behavior in Drosophila flies is mediated by pheromones that can act in concert with odors of the habitat of the flies and indicate a suitable habitat for mating and oviposition (Bartelt et a!., 1986; Scha­ ner et a!., 1987). An aggregated distribution may reduce interspecific compe­ tition and facilitate coexistence between species (Atkinson and Shorrocks, 1981; rves, 1988), but should also be important for finding mates. Drosophila aggre­ gation pheromones are produced by males and attract flies of both sexes (Bartelt and Jackson, 1984; Bartelt et aL 1985; Jaenike et a!., 1992). The pheromones are transferred to females during mating and transferred further to an oviposition site by female flies (Bartelt et a!., 1985). It has been suggested that these pheromones have functions in addition to aggregation behaviors, such as pre­ venting matings of newly mated females and interactions between males (Zawis­ towski and Richmond, 1986). Aggregation pheromones ofDrosophila are generally volatile esters, ketones or unsaturated hydrocarbons (Bartelt et a!., 1985, 1988; Moats et a!., 1987). Several species produce the same compound; for instance, species of the Dro­ sophila melanogaster group all have (Z)-II-octadecenyl acetate as an aggre­ gation pheromone (Bartelt et a!., 1985; Schaner et a!., 1987, 1989). Such nonspecific pheromones can cause interspecific aggregates of related flies in a breeding habitat (Jaenike et a!., 1992). However, flies also have a range of cuticular hydrocarbons that are assumed to take part in regulating mating behav­ iors (Scott et aL 1988; Cobb and Jallon, 1990). A mixture of these compounds may give species specificity at close range. Apart from increasing the risk of intraspecific competition, aggregation pheromones can also affect the risk of parasitization as parasitoid wasps are attracted to the pheromones of adult Drosophila flies when searching for their larval hosts (Wiskerke et a!., 1993). However, the risk of an individual Dro­ sophila larvae to be attacked by a parasitoid should decrease at increasing den­ sities of flies according to the functional response of the parasitoid (Hertlein and Thorarinsson, 1987). Recently a study on parasitoid wasps showed new and unexpected results when both generalist and specialist larval parasitoids of dro­ sophilids were attracted to aggregation pheromones of adults of some host as well as some nonhost species (Hedlund et a!., 1996). To interpret the results of the latter study, the aggregation pheromones of the Drosophila species involved need to be chemically identified. From the study of parasitoid-host interactions (Hedlund et a!., 1996) we have chosen three species, Drosophila phalerata (Meigen) and Drosophila subobscura (Collin) with a European distribution and one cosmopolitan species, Drosophila immigrans (Sturtevant) (Wheeler, 1981). This study aims at iden­ tifying aggregation pheromones of these three species not only to increase the knowledge of communication of Drosophila but also that of parasitoid-host interactions. AGGREGATION PHEROMONES 1837

METHODS AND MATERIALS

Fly Cultures Drosophila immigrans was reared on a medium ofapple, water, agar, sugar and yeast. The culture originated from flies reared out of fruits collected in orchards in The Netherlands. D. subobscura was reared in the same way as D. immigrans. The culture was established from flies reared from sap fluxes of trees originating from The Netherlands. D. phalerata was reared on decaying Agaricus hortensis mushrooms. The culture originated from flies reared out of Phallus impudicus mushrooms col­ lected in The Netherlands. The flies were reared in 250 ml glass vials at 20°C on a 16: 8 (L: D) photocycle. pupae were washed out of the vial and transferred to 200 ml glass vials containing a layer of water agar. Fly Extracts. Flies were separated by sex 0-6 hr after emergence from the pupae and transferred to new glass vials with a layer of water agar and honey on a strip of filter paper. To get sexually immature and mature flies the flies were collected at day one and day seven, respectively, and soaked in hexane (l00 flies ml- I hexane) at room temperature. The crude extracts were removed after 24 hr and kept at -20°C until further analysis. GC-MS Analysis. The crude extracts were analyzed with gas chromatog­ raphy (GC) and mass spectrometry (MS). Male- and female-derived extracts were compared by capillary gas chromatography (DB-l column, 15 m length, 1 f.Lm film thickness, 0.25 mm ID; temperature programmed from 50°C to 300°C at lOoC per min, with a final hold of 10 min at 300°C). GC retention indices relative to n-alkanes were calculated for sex-specific compounds and other, relatively abundant compounds. The Hewlett Packard 5890 gas chro­ matograph was equipped with splitless injector, flame ionization detector, and Hewlett Packard 3396A integrator. Electron impact mass spectra were obtained for the detected sex-specific compounds on a Hewlett Packard 5970 Mass Selec­ tive Detector with capillary GC inlet. Additional analyses were performed after removal of fatty acids by washing samples with 5 % (w: w) Na2C03 solution, since the large broad, fatty acid GC peaks could have obscured minor sex­ specific compounds. Saturated hydrocarbons were identified from mass spectra and GC retention indices. Unsaturated hydrocarbons and esters were identified by mass spectra, GC retentions, and mass spectra of dimethyldisulfide (DMDS) adducts (Carlson et aI., 1989). The DMDS derivatives allowed location of double bonds, and double bond configuration was determined by GC retention in comparison with authentic standards. Sex-specific compounds were considered as possible pher- 1838 HEDLUND ET AL. omone components. When possible, synthetic versions of the sex-specific com­ pounds were evaluated in the bioassay to verify behavioral activity. Synthetic Compounds. (Z)-ll-octadecenyl acetate, also called cis-vaccenyl acetate (cVA) was purchased from Sigma (St Louis, USA). The 5-tricosenes were prepared by the method of Sonnet (1974) from pentanal and (octade­ cyl)triphenylphosphonium bromide, and the Z and E isomers were separated by AgNOrHPLC as described by Heath and Sonnet (1980). Bioassays. All bioassays were done in a cage (width 30 cm, depth 40 cm, height 35 cm) with nylon mesh (0.1 mm) along its long sides. A fan outside the cage generated an air How of 0.1 m S-I inside the cage. Each cage was stocked with 300 flies 16 hr before the tests. The flies were two to nine days old and of equal sex ratio. They were left with honey and water until the tests started. Fly extracts or synthetic compounds were applied to a filter paper (1 cm x 6 cm strip). The solvent was allowed to evaporate and the filter paper was inserted into a small glass vial (5.5 cm x 1.5 cm diameter). A drop of water was applied on the bottom of the vial. When a food substrate was present in the vial, either a 5 mm layer of an apple-yeast mixture (15: 1) (w: w) or pieces of decaying mushrooms (Agaricus hortensis) were placed at the bottom of the vial. In each test two vials were placed inside the cage and perpendicularly to the air flow. One vial contained a hexane control and the other an extract or a synthetic compound dissolved in hexane. Tests with D. phalerata and D. subobscura lasted for 5 min while tests with D. immigrans lasted for 10 min. During this period of time the flies were allowed to enter a vial. At the end of the test the vials were capped with a cotton plug. The number and sex of the flies were determined. Subsequent tests separated by 3 to 5 min between the tests were done with the same cage. The cage was stocked with fresh flies each experimental day. All tests were performed at 20°C. Each treatment (female extract, male extract etc.) was replicated 10 to 20 times and a series of treatments was carried out each experimental day (block design) so that any variation due to daily activities of the flies could be elimi­ nated. A treatment was compared to its control with a Wilcoxon two sample test (Sokal and Rohlf, 1981) and a Kruskal-Wallis (Hollander and Wolfe, 1973) test compared the number of flies in traps of different treatments.

RESULTS

Hexane extracts of mature male and female D. immigrans were attractive to both sexes (52 % males, 48 % females) when responses were compared to a hexane control (Table 1). However, more than twice as many flies responded to male extracts as to female extracts. The GC-MS analysis revealed that extracts of mature males contained small amounts (30 ng/male fly) of (Z)-ll-hexade- AGGREGATION PHEROMONES 1839

TABLE 1. BIOASSAY ACTIVITY (MEAN No OF FLIES CAPTURED IN VIALS) OF Drosophila immigrans AND D. phalerata IN A WINDTUNNEL BIOASSAY TO 7.5 J.tg SYNTHETIC cVA ((2)-11­ OCTADECENYL ACETATE) AND CRUDE FLY EXTRACTS (IN EQUIVALENTS OF TEN FLIES)

Treatment Bioassay activity

pheromone control pheromone control mean" (sd) mean" (sd) ph N

D. immigrans female extract hexane I. lOa (1.85) O.IOa (0.32) 0.05 10 male extract hexane 2.80b (2.30) O.IOa (0.32) 0.001 10 cVA hexane I. 15a (0.75) 0.20a (0041) 0.001 20 cVA & apple yeast hexane & apple yeast 5.33b (4.58) 1.14b(1.0l) 0.001 20 D. phalerata female extract hexane I. 15a (I. 12) 0.68a (0.95) n.s. 20 male extract hexane 8.15b (3.44) 0.75a (I.07) 0.001 20 cVA hexane 2AOa (1.23) 0.55a (0.76) 0.001 20 cVA & mushroom hexane & mushroom 3AOa (2.50) UOa (1.44) 0.01 20

"Means followed by different letters are significantly different at the 0.05 level according to multi comparison tests on Kruskal-Wallis rank sums (Hollander and Wolfe. 1973). h Differences between pheromone treatment and the control treatment according to a Wilcoxon two sample test (Sokal and Rohlf. 1981).

cenyl acetate and large amounts (750 ng/male) of (Z)-octadecenyl actetate, i.e., cVA (Tables 2, 3). Both sexes also produced 2-methyloctacosane and 2-meth­ yltriacontane. When synthetic cVA was presented in the bioassay, flies were attracted to cVA independent of sex (50% males, 50% females) (Table 1). A combination of cVA and food (apple and yeast) increased the number of flies attracted to cVA by about five times compared to tests based on cVA only. However, the proportions of flies attracted to cVA vs. control were the same in both cases. Bioassays of hexane extracts of D. phalerata showed that flies of both sexes (53% males, 47% females) were attracted to adult male extracts (Table 1). The same compounds were identified in these extracts as in those produced by D. immigrans, i.e., (Z)-ll-hexadecenyl acetate and cVA (Tables 2,3). D. phalerata flies were also attracted to cVA in the bioassay (Table 1). However, when cVA was tested in combination with decaying mushrooms the response of the flies did not increase. The number of D. phalerata flies attracted to extracts of males was about two to three times higher than when cVA was tested alone or in combination with mushroom. D. subobscura flies of both sexes (52 % males, 48 % females) were attracted to hexane extracts of male flies, but only when the extracts were applied on 1840 HEDLUND ET AL.

TABLE 2. COMPOUNDS IDENTIFIED IN EXTRACTS OF ONE AND SEVEN DAYS OLD FLIES OF Drosophila immigrans AND Drosophila phalerma

Amounr (nglfty)

Compound Producing sex 7 days I day

D. immigrans (Z)-ll-hexadecenyl acetate male 30 (Z)-ll-octadecenyl acetate male 750 180 2-methyloctacosane male & female 240 100 2-methyltriacontane male & female 240 200 D. phalerata 30 (Z )-1 l-hexadecenyI acetate male 30 (Z)-Il-octadecenyl acetate male 750 80 2-methyloctacosane male & female 150 200 2-methyltriacontane male & female 120 150

food (apple and yeast) (Table 4). Identification of compounds in the extracts showed that mature male flies produced about 30 ng cVAlfly and 120 ng (Z)­ 5-tricosenelfly (Tables 3, 5). Both sexes of D. subobscura produced large amounts of 5,9-pentacosadiene and minor amounts of 5-pentacosene, 2-meth­ y1hexacosane and 5,9-heptacosadiene. Male and female D. subobscura produced 2-methyloctacosane and 2-methyltriacontane as did the other two Drosophila species. The male specific compounds, cVA and (Z) or (E)-tricosene, did not

TABLE 3. THE KEY MS IONS (M/Z) OF UNDERIVATIZED AND DERIVATIZED FLY EXTRACTS OF THE IDENTIFIED COMPOUNDS. GC RETENTION INDEX Is RELATIVE TO A STANDARD WITH n-ALKANES

GC retention Key MS ions Key MS ions for DMDS Compound index (//lIz) derivatives (//lIz)

(Zl-Il-octadecenyl acetate 2166 310. 250 404. 145. 259 (Zl-Il-hexadecenyl acetate 1974 282. 222 117.259. (Z)-5-tricosene 2291 322 416. 117.299 (Z)-5-pentacosene 2492 350 444. 117.327 5.9-pentacosadiene 2476 348 (mono-DMDS adduct) 442.117.325.171. 271 5.9-heptacosadiene 2679 376 (mono-DMDS adduct) 470.117.353.299 2-methylhexacosane 2665 380. 365. 337 2-methyloctacosane 2866 408. 393. 365 2-methyltriaconrane 3066 436. 42 I. 393 AGGREGATION PHEROMONES 1841

TABLE 4. BIOASSAY ACTIVITY (MEAN No OF FLIES CAPTURED IN VIALS) OF Drosophila slibobsclira TO CRUDE FLY EXTRACTS (TEN FLY EQUIVALENTS) AND SYNTHETIC (2) AND (E)­ TRICOSENE AND cVA, (2)-11-0CTADECENYL ACETATE

Treatment Bioassay activity

pheromone control pheromone control mean" (sd) mean" (sd) ph N male extract hexane Oo4Oa (0.74) Oo4Oa (0.63) n.s. 15 male extract & apple yeast hexane & apple yeast 4.13b (2.17) 2047b (1.88) 0.05 15 female extract hexane O.lla (0.32) O.27a (0046) n.s. 18 female extract & apple yeast hexane & apple yeast 4.38b (4.01) 4.62b (4.59) n.s. 13 (Z)-tricosene (1.2 Jlg) hexane 0.07a (0.26) 0.07a (0.26) n.s. 15 (£)-tricosene (1.2 Jlg) hexane O.27a (0.59) O.27a (0.59) n.s. 15 cVA (3.0 Jlg) hexane 0.13a (0.35) Oo4Oa (0.51) n.s. 15

"Means followed by different letters are significantly different at the 0.05 level according to multi comparison tests on Kruskal-Wallis rank sums (Hollander and Wolfe. 1973). h Differences between the pheromone treatment and the control treatment according to a Wilcoxon two sample test (Sokal and Rohlf. 1981).

attract flies in the windtunnel bioassay (Table 4). However. as these compounds were not tested in combination with food, further experiments are needed to reveal the attractivity of the compounds together with a food substrate.

DISCUSSION

The aim of this study was to identify the aggregation pheromones of the three Drosophila species involved. Both males of D. immigrans and males of

TABLE 5. COMPOUNDS IDENTIFIED IN HEXANE EXTRACTS OF SEVEN DAYS OLD Drosophila slibobsclira

Identified compounds Producing sex Amount (nglfly)

(Z)-II-octadecenyl acetate (cVA) male 30 (Z)-5-tricosene male 120 5.9-pentacosadiene male & female 1000 (Z)-5-pentacosene male & female 200 2-methylhexacosane male & female 90 5.9-heptacosadiene male & female 120 2-methyloctacosane male & female 250 2-methyltriacontane male & female 25 1842 HEDLUND ET AL.

D. phalerata produce (Z)-ll-octadecenyl acetate (cVA) as a major pheromone component. In both species extracts of male flies as well as synthetic cVA attract male and female flies, and we conclude that cVA is an aggregation pheromone compound. However, male extracts of D. immigrans attract twice as many flies as pure cVA, and in the case of D. phalerata male extracts attract almost four times as many flies. The pattern of higher attractivity to extracts than to pure cVA has been shown for other cVA-producing species such as D. melanogaster (Bartelt et aI., 1985), whereas other species, such as D. simulans, show no difference in attractivity between fly extracts and synthetic cVA (Schaner et aI., 1987). A higher level of attractivity to fly extracts than to pure cVA, or to female extracts as in D. immigrans, may indicate that there are other compounds present in the extracts that are active in eliciting aggregative behaviors of the flies. Other compounds may be produced in small amounts by both sexes but have to act in concert with cVA to attract flies. (Z)-11-hexadecenyl acetate that was found as a male specific compound of D. immigrans and D. phalerata is produced in small amounts and may be a possible coattractant. Although further work is needed to test the activity of (Z)-hexadecenyl acetate and chemically identify other active compounds, cVA can be regarded as a major aggregation pheromone of D. immigrans and D. phalerata. Aggregative behavior of D. subobscura was only observed towards male fly extracts in combination with food of the flies. To identify the main aggre­ gation pheromone compound(s) of D. subobscura further work is needed with testing attractivity of flies to combinations of identified compounds and food, and fractionation of the extracts. However, the identified hydrocarbons reveal a system quite different from that of D. immigrans and D. phalerata. Rather it has similarities with the aggregation pheromones of the D. virilis subgroup in which a combination of hydrocarbons and male specific compounds elicits aggre­ gative behaviors (Bartelt et aI., 1986). The presence of food odors seems to be necessary for D. subobscura to respond to a pheromone whereas it increases the attractivity of cVA for D. immigrans. Coattraction of flies to food odors and aggregation pheromones may be a general pattern among many Drosophila species. The attractivity of aggre­ gation pheromones such as cVA have been shown to increase in combination with food substrates for several fly species of the melanogaster species group (Schaner et aI., 1987; 1989). Other types of pheromone compounds, e.g., tig­ lates of flies of the virilis species group, can also be synergistic in combination with food odors (Bartelt et aI., 1986). cVA, the aggregation pheromone of D. immigrans and D. phalerata, is produced by a number of species within the subgenus Drosophila and the subgenus Sophophora (Bartelt et aI., 1985; Schaner et aI., 1987, 1989; Jaenike et aI., 1992). The risk of increasing interspecific competition that follows upon the use of the same aggregation pheromone compound may be reduced in nature AGGREGATION PHEROMONES 1843 by different usage of microhabitats. Of the three species studied here only two produce cVA as the major pheromone compound and of these D. immigrans breeds mainly in fermenting fruit (Atkinson and Shorrocks, 1977), whereas D. phalerata is a woodland species that breeds in decaying mushrooms (Janssen et al., 1988). The attraction of flies to a combination offood odors and aggregation pheromones, as discussed above, will probably decrease the risk of competition between the species. However, fly species with similar aggregation pheromones and breeding sites have been shown to form interspecific aggregations (Jaenike et aI., 1992).

Acknowledglllems-KH's stay in The Netherlands was founded by The Swedish Forestry and Agricultural Research Council. and The Swedish Institute. We thank Henk Snellen for rearing the .

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