690 9

ECOLOGY AND POPULATION BIOLOGY Aggregations in Mycophagous (Diptera: ): Candidate Pheromones and Field Responses

JOHN JAENIKE, ROBERT J. BARTELT,l ANDREA F. HUBERTY, STEPHEN THIBAULT,2 AND JEFFREY S. LIBLER3 Department of Biology, University of Rochester, Rochester, NY 14627

Ann. Entomol. Soc. Am. 85(6): 696-704 (1992) ABSTRACT Hexane extracts from Drosophila falleni Wheeler, D. recens Wheeler, D. putrida Sturtevant, and D. testacea Roser contained a variety of compounds that, like compounds known as aggregation pheromones in other species of Drosophila, are pro­ duced only by mature males. Application of crude hexane extracts to in the field significantly increased the numbers of Drosophila spp. captured at the mushrooms. We conclude that these volatile compounds can influence the distribution of across breeding sites in the field.

KEY WORDS aggregation pheromones, behavior, cuticular hydrocarbons

INSECTS THAT USE PATCHY, ephemeral re­ site, and aggregations of ovipositing females sources such as fruits, fungi, dung, and carrion (Jaenike & James 1991). are frequently aggregated across breeding sites Here, we are concerned with possible causes both as adults and larvae (Hanski 1987; Ives of aggregations of breeding adults across re­ 1988a, 1991; Rosewell et al. 1991). Such aggre­ source patches, a subject that has so far received gations have been recognized as important for little attention from field ecologists. There are the structure of these communities be­ several possibilities. First, breeding sites located cause they promote the coexistence of ecologi­ in the more favorable microhabitats are visited cally similar but independently distributed spe­ by greater numbers of breeding adults. For ex­ cies (Atkinson & Shorrocks 1981, Ives 1988b). ample, Hanski (1987) found that position within This is because intraspecific aggregations am­ a pasture can affect the distribution ofdung bee­ plify levels of intraspecific competition, leaving tles. some resource patches underused and thus avail­ Second, the intrinsic properties of resource able for competing species. patches vary, such that some are more attractive Field studies of mycophagous species of than others. For instance, Ives (1991) found that Drosophila have demonstrated that larvae are carrion flies were consistently attracted to partic­ frequently food-limited and thus subject to both ular carcasses regardless oftheir spatial position. intra- and interspecific competition (Grimaldi & Variation in microhabitat and resource quality Jaenike 1984; A. C. James, personal communica­ probably contribute to aggregations in most spe­ tion). As in other that use patchy re­ cies of insects using patchy resources. sources, the coexistence of several ecologically Third, some sort of positive feedback occurs, similar species of these Drosophila is facilitated the insects being preferentially attracted to by aggregated distributions oftheir larvae across breeding sites that have already been visited by mushrooms. These intraspecific aggregations of other insects. Attraction of adult flies to odors larvae are caused by two factors: individual fe­ associated with larvae is one possibility (Hoff­ males laying more than one egg at a breeding mann & Parsons 1986). Aggregation phero­ mones, the subject of the present study, are an­ other. These pheromones have been discovered

1 USDA-ARS National Center for Agricultural Utilization in several species of Drosophila and their effec­ Research, Bioactive Constituents Research, 1815 North Uni­ tiveness verified in wind tunnel assays (e.g., versity Street, Peoria, IL 61604. Bartelt et al. 1985b, 1986a). All aggregation pher­ 2 Department of Biology, Wabash College, Crawfordsville, omones previously identified in other Droso­ IN 47933. 3 Department of Biology, DePauw University, Greencastle, phila spp. are produced by mature males but IN 46135. attract flies of both sexes. The pheromones fall

0013-8746/92/0696-0704$02.00/0 © 1992 Entomological Society of America November 1992 JAENIKE ET AL.: AGGREGATIONS OF MYCOPHAGOUS DROSOPHILA SPP. 697 into three general classes: (1) hydrocarbons of flies had been separated by sex shortly after eclo­ the cuticular surface, (2) more polar lipids (e.g., sion and thus were virgin. Extracts were kept at ketones and esters of 13-22 carbon atoms) origi­ -80°C before analysis. nating in the reproductive tract, and (3) very vol­ The constituents of the extracts were identi­ atile esters (e.g., 6-8 carbon atoms) of uncertain fied through the use ofchromatography and mass origin (e.g., Bartelt et al. 1986b, 1989; Schaner et spectrometry. An aliquot ofeach extract was sub­ al. 1987). These pheromones often act synergis­ jected to column chromatography on silica gel tically with volatile compounds emanating from (0.5 x 2.5-cm column) to separate the compo­ the resource itself to enhance attraction (Bartelt nents by polarity. The elution solvents were hex­ et al. 1986a, 1988). However, several factors, in­ ane and 5, 10, and 50% ether in hexane (two cluding turbulence (Elkinton et al. 1987), the column volumes per solvent): hydrocarbons distance between flies and their resources, and eluted with hexane; esters, with 5% ether in general environmental complexity, may hamper hexane; and primary alcohols, with 50% ether the influence ofsuch aggregation pheromones in in hexane. Fractions were analyzed further by the field. In fact, the operation of such phero­ gas chromatography (GC), and the chromato­ mones in a field setting has not yet been demon­ grams were compared to determine whether sex­ strated in any species of Drosophila. specific or age-specific compounds existed and to The present study focuses on aggregations in confirm that the major constituents were still common species of mycophagous Drosophila of present in at least one ofthe silica gel fractions. the northeastern United States: D. falleni Free fatty acids were usually removed from the Wheeler and D. recens Wheeler of the quinaria crude extracts before GC analysis was done by species group and D. putrida Sturtevant and D. washing the samples with 5% Na2C03 solution. testacea Roser of the testacea group. All these Removal offree acids, which were present in all species exhibit strong intraspecific aggregations samples, allowed other constituents of similar which are greatest for species belonging to the retention times to be observed more easily. same species group and are weaker, but often A Hewlett Packard 5890 gas chromatograph, significantly positive, for species belonging to equipped with a flame ionization detector and different groups (Jaenike & James 1991). splitless capillary injector and interfaced to a We investigated whether any chemicals pro­ Spectra-Physics SP4400 integrator, was used. duced by these species ofDrosophila are consis­ The carrier gas was helium. The column was a tent with the observed patterns of intra- and in­ Durabond DB-l capillary (15 m long, 0.25 mm terspecific aggregation and experimentally inside diameter, 0.25-pm film thickness). The tested whether crude extracts from these flies temperature program was usually 100-320°C at can bring about aggregations under field condi­ lOoC per minute, except that the program for tions. Because the chemistry of known Droso­ ester analyses began at 40°C. Kovats retention phila pheromones is diverse, a broad range of indices (KI) relative to n-alkanes were calculated compounds from the four subject species was by linear interpolation (KI for n-alkanes is de­ analyzed, so that sex-specific pheromone candi­ fined to be 100 times the number of carbons). dates in any of the known classes would not be The retention indices were especially useful in overlooked. Structures of several such com­ the analysis of hydrocarbons (see Bartelt et al. pounds were established. 1986b). Compounds were quantified on a per- basis by using integrator peak areas relative to an external standard (heptadecane). Materials and Methods Electron impact mass spectra (MS) were ob­ Chemical Analyses. Volatile compounds from tained for all extract components down to =1 ng D. falleni, D. recens, D. putrida, and D. testacea per fly-equivalent on a Hewlett Packard 5970 in­ were examined. All four species are members of strument which was interfaced to a gas chromato­ the immigrans-Hirtodrosophila radiation of the graph and column identical to those described subgenus Drosophila (Throckmorton 1975). above. The MS, KI, and polarity-functional group Each strain was established from 8-10 insemi­ data were sufficient to identify completely many nated females collected near Rochester, NY, in compounds in the extracts. A mass spectral library 1990. These species were maintained in the lab­ and a series ofauthentic chemical standards aided oratory at 22°C on Instant Drosophila Medium in these efforts. Key mass spectral ions are listed (Carolina Biological Supply Company, Burling­ in the tables ofresults along with compound iden­ ton, NC) plus a piece of commercial , tifications; some low-intensity ions were not ob­ bisporus Lange. served for minor constituents of the extracts. Compounds were extracted by soaking whole Unsaturated esters were derivatized with di­ flies in hexane (100 flies per ml) for 24 h. Extrac­ methyl disulfide (DMDS) and analyzed by MS to tions were carried out separately on immature determine locations of double bonds (Carlson et and mature males and females. Immature flies al. 1989). Double-bond configurations in the un­ were <2 h post-eclosion, whereas mature flies derivatized samples were determined from GC had been kept as adults at 22°C for 7 d. Mature retentions by comparison to standards. 698 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 85, no. 6

Field Behavioral Assays. Hexane extracts were significant differences in the proportions of the obtained as described above from mature adults two species groups as follows: a significant inter­ ofD. falleni and D. putrida; these were stored at cept would indicate that one species group gen­ -20°C until ready for use. Four field trials were erally outnumbered the other across trials. A sig­ carried out in July and August 1991, two each at nificant trial effect would show that the relative Brighton Town Park and Powder Mill Park, both proportions of the two species groups varied in Monroe County, NY. For trial 1 at Brighton among the four field trials. A significant bait Town Park, the hexane extracts were obtained within trial effect would indicate that, taking all solely from male flies. In the remaining three four trials into consideration, the relative propor­ trials, extracts obtained from males and females tions ofthe two species groups differed between were pooled. In each trial, mushroom baits (com­ the D. falleni- and the D. putrida-treated baits. mercial Agaricus bisporus) were set out at 8-10 Note on Systematics. The species referred to sites; the sites were separated by at least 20 m. At as in this paper has recently each site, three piles of five mushrooms each been described as were placed on the ground; the different piles at (Grimaldi, D., A. C. James, and J. Jaenike. Sys­ each site were separated by at least 2 m. A piece tematics and modes of reproductive isolation in of filter paper was placed on each pile, and we the Holarctic Drosophila testacea species group applied either 0.5 ml pure hexane (control treat­ (Diptera: Drosophilidae). Ann. Entomol. Soc. ment), 0.5 ml ofhexane extract from D. falleni, or Arner., in press.) 0.5 ml of hexane extract from D. putrida to this paper each evening during the course of a trial. Assignment of the treatments was determined Results randomly (by coin tosses) after the mushrooms Chemical Analysis. Chemical comparisons re­ had been set out. Flies were collected by sweep vealed dramatic differences in ester and alcohol netting several times per day for the next 3-4 d, profiles between sexually mature males and all a period during which the mushrooms remained other flies (Fig. 1). Several compounds specific to attractive to flies. mature males were identified (Table 1). In all Analysis of Field Data. Because of a drought four species, the mature males contained rela­ during much ofthe summer of1991 in the Roch­ tively massive amounts (200-770 ng) of (Z)-l1­ ester area, Drosophila population densities were octadecenyl acetate (also known as cis-vaccenyl consistently very low. As a result, the individual acetate) and minor amounts of the acetates mushroom baits attracted very few flies. There­ of the homologous 16- and 20-carbon alcohols. fore, for purposes of analysis, we pooled the fly Mature males of D. testacea also contained (Z)­ numbers for all ofthe mushroom baits ofa given 9-octadecenyl acetate (oleyl acetate). Other es­ type for each trial. In the statistical tests, each ters were detected in minor amounts but could trial is treated as one replicate. Furthermore, be­ not be fully characterized (Table 1). In addition, cause of the low capture numbers, we have not ethyl hexanoate was identified in all four species attempted to distinguish between the attractive­ and isopropyl hexanoate from D. putrida and D. ness of extracts from a given species (e.g., D. falleni. Finally, there was one alcohol detected putrida) to conspecifics and to other species of from all species, (Z)-11-octadecen-1-01 (the ace­ the same species group (e.g., D. testacea). We tate of this alcohol was the major ester in the have pooled capture numbers for flies belonging flies.) All these compounds were detected only to the same species group. We were concerned in males, and then only when they were sexually with two questions: (1) Were more flies captured mature. at baits treated with fly extracts than at the con­ There were some differences in the profiles of trol baits? To address this question, the total male-specific compounds among the four species numbers ofthe four Drosophila species captured (Table 1; Fig. 2). In D. putrida and D. testacea, at the control baits were compared with the num­ (Z)-l1-eicosenyl acetate was more abundant bers caught at the extract-treated baits. We used than (Z)-l1-hexadecenyl acetate, and in D. fal­ a nonparametric test, which is based on the null leni and D. recens, the reverse was true. In the hypothesis that within a given trial, there is a testacea group species, (Z)-11-octadecen-1-01 one-third chance that the fewest flies will be and (Z)-l1-eicosenyl acetate were about equally caught at the control baits. (2) Did the relative abundant, whereas in the quinaria group spe­ numbers of captured flies belonging the D. tes­ cies, the former compound was considerably tacea and D. quinaria species groups depend on more abundant. Only D. testacea contained (Z)­ the species from which the hexane extracts were 9-octadecenyl acetate. obtained? Here, numbers offlies ofeither the D. Less volatile compounds (e.g., >30 carbon at­ quinaria or the D. testacea group captured at oms) do exist that are specific to mature males either D.falleni- or D. putrida-treated baits were and that differ among species (Table 1; Fig. 2). pooled over all baits and all collections within These compounds are more polar than hydrocar­ each ofthe four trials. The CATMOD procedure bons, but the structural details remain undeter­ (SAS Institute 1985) obtains :Jf values to test for mined. November 1992 JAENIKE ET AL.: AGGREGATIONS OF MYCOPHAGOUS DROSOPHILA SPP. 699

MATURE MALE

o o o o o CD I'- C1> I') N

MATURE FEMALE

NEWLY EMERGED MALE

NEWLY EMERGED FEMALE

I 5 10 15 20 RETENTION TIME (MIN)

Fig. 1. Gas chromatograms illustrating chemical differences as a function of sex and age in D. falleni. GC program was l00-320°C at 10°C/min. Kovats retention indices are listed above selected peaks; (H) indicates that the peak is a hydrocarbon. Chromatograms represent crude fly extracts from which fatty acids had been removed with Na2C03'

In contrast, no cuticular hydrocarbons fitted than with the esters. Most important, there was the pattern expected for aggregation pheromone no qualitative male-female dimorphism de­ components. All of the flies, regardless of spe­ tected in the hydrocarbon profiles ofany species. cies, sex, or age, contained n-alkanes of17 and 19 Finally, every extract contained the following carbons and 2-methylalkanes of 29 and 31 total free fatty acids: oleic, linoleic, palmitic, myristic, carbons (Table 2). In addition, newly emerged capric, and mono-unsaturated acids of16, 14, and flies also contained small amounts ofunsaturated 12 carbon atoms. The total amounts of acids var­ hydrocarbons such as 7-nonacosene or the tritri­ ied considerably among extracts, probably be­ acontadienes. Total amounts of hydrocarbon did cause the acids were located deep within the increase with age, and certain alkenes and alka­ flies and were not removed by the hexane soaks dienes were present in mature flies which were with uniform efficiency. The 18- and 16-carbon not present in newly emerged flies (e.g., 9­ acids were always the most abundant. It is un­ nonacosene and a hentriacontadiene in D. fal­ known whether these acids were normally leni), but changes with age were far more subtle present in the free form or whether they were 700 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 85, no. 6

Table I. Compounds found only in extracts of sexually mature male flies (amounts in ng per fly) Kovats Silica gel index D. putrida D. testacea D. recens D·falleni fraction" Identification (key MS ions, rnJz)b 0981 0.24 0.09 0.17 0.20 5% ether Ethyl hexanoate (144, 101, 99, 88) 1023 0.04 0.19 5% ether Isopropyl hexanoate (117, 101,99,87) 1974 2.0 2.1 8.5 7.2 5% ether (Z)-l1-hexadecenyl acetate (282,222; DMDS: 259, 117) 2039 8.0 4.4 23.0 35.0 50% ether (Z)-ll-octadecen-l-ol (268,250) 2161 27.0 5% ether (Z)-9-octadecenyl acetate (310,250; DMDS: 231, 173) 2166 200.0 270.0 570.0 770.0 5% ether (Z)-ll-octadecenyl acetate (310,250; DMDS: 259, 145,404) 2352 0.9 2.3 0.8 5% ether (undetermined) 2360 9.3 11.0 7.1 5% ether (Z)-l1-eicosenyl acetate (278; DMDS: 259, 173) 2367 1.3 7.5 1.4 5% ether (undetermined) 2561 2.1 5% ether (undetermined) 2806 3.0 3.3 4.6 5% & 10% ether (undetermined) 3310 42.0 10% ether (undetermined) 3325 8.8 10% ether (undetermined) 3498 30.0 10% ether (undetermined) 3507 4.0 10% ether (undetermined) 2::3523 >50.0 2::12.0 2::13.0 >20.0 5% or 10% ether, (undetermined) or both " Percent ether in ether-hexane solvent which eluted the compound from silica gel. b Key ions observed. Some expected, low-intensity ions were not seen for some minor components. DMDS, key ions from dimethyl disulfide adducts. released by postmortem degradation of neutral bait types within trials (P = 0.014). Relatively fats. Because these chemicals were present in all more flies of the D. quinaria group were at­ of the extracts, it is suggested that they have no tracted to the D. falleni-treated baits, whereas relevance in the aggregation characteristics of the D. testacea group flies were relatively more these flies. The fatty acids produce broad GC commonly captured at the D. putrida-treated peaks and could interfere with the analysis of baits. The CATMOD analysis revealed that, extracts, but the acids can be removed with within trials, the effect ofbait type on these pro­ Na2C03 (Fig. 3). portions was significant at the 0.05 level for the Field Data. The numbers offlies ofeach ofthe Brighton (July) and Powder Mill (August) collec­ four species captured at each type of bait in the tions and was marginally significant for the Pow­ four field trials are shown in Table 3. Over the der Mill (July) collection (P = 0.07). In the Brigh­ four trials, an average of =70% more flies was ton (July) trial, the hexane extracts were obtained captured at the baits treated with extracts of ei­ solely from mature male flies. ther D. falleni or D. putrida than at the control baits. In each of the four trials, fewer flies were Discussion captured at the control baits than at either the D. falleni- or D. putrida-treated baits. We thus re­ Our study was designed to examine the possi­ ject the null hypothesis that the three types of ble connection between fly-derived chemicals baits are equally attractive to these Drosophila and patterns of aggregation observed in natural (P = (1/3)4 = 0.012). populations ofmycophagous Drosophila spp. We The CATMOD analysis ofthe numbers offlies focused on the four common species ofthe north­ belonging to either the D. quinaria and D. tes­ eastern United States: D. falleni and D. recens of tacea group captured at the two types of phero­ the quinaria species group and D. putrida and mone-treated baits is shown in Table 4. The J? D. testacea ofthe testacea group. The most strik­ value associated with the intercept in the ing patterns previously observed in collections CATMOD analysis indicates that there was a ofnaturally occurring mushrooms include aggre­ marginally significant tendency for the D. qui­ gations of conspecific adults across mushrooms naria group to outnumber the D. testacea group among all four species, strong correlations be­ within trials (P = 0.06). However, the relative tween species belonging to the same species proportions of the D. quinaria and D. testacea group, and weak but positive correlations be­ groups did vary significantly among the four tri­ tween species belonging to different groups als (P < 0.0001). Finally, and most important, the (Jaenike & James 1991). relative proportions of the two species groups Our field studies show that volatile com­ differed consistently and significantly among pounds extracted from these flies can influence November 1992 JAENIKE ET AL.: AGGREGATIONS OF MYCOPHAGOUS DROSOPHILA SPP. 701

D. putrida

.--. J: ~

If) :c <0 ~ 0 I') <0 :c .--. <0 ~ J: co 0 N <0 0> If) I') <0 I') N o o ' 0>

D. testacea

D. recens

D. falleni

I 5 10 15 20 RETENTION TIME (MIN)

Fig. 2. Gas chromatographic comparison of mature males of four species. GC program was I00-320°C at IO°C/min. Kovats retention indices are listed above selected peaks; (H) indicates that the peak is a hydrocarbon. Fatty acids were removed before GC with NaZC03 • the distribution of adults ofthe four Drosophila age. The major compound in our work, (Z)-l1­ spp. across mushrooms. Mushrooms treated with octadecenyl acetate, has been reported previ­ these chemical mixtures attracted significantly ously as an aggregation pheromone in three other more flies in total than did the control mush­ Drosophila species: D. melanogaster Meigen, D. rooms. Most notably, baits treated with D. falleni simulans Sturtevant, and D. ananassae Dole­ extracts attracted relatively more flies of the D. schall. The minor homolog, (Z)-l1-eicosenyl ace­ quinaria species group, whereas those treated tate, was also active in D. ananassae and in D. with extracts from D. putrida were relatively bipectinata Duda and D. malerkotliana Parshad more attractive to flies of the D. testacea group. & Paika as well (Schaner et al. 1987, 1989a, b; The chemical patterns observed in our re­ Bartelt et al. 1985a). However, this is the first search support the idea that aggregation phero­ report ofthese esters in the subgenus Drosophila; mones exist in these species. As with other all the other species belong to the subgenus Drosophila spp. in which aggregation phero­ Sophophora. mones have been studied, the mature males Another male-specific compound in the four possess volatile compounds that are lacking mycophagous species considered in this study in newly emerged males and in females of any has been found before in the subgenus Droso- 702 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 85, no. 6

Table 2. Major cuticular hydrocarhons from four Drosophila species and comparisons with respect to sex and age (amounts in ng per fly)

Kovats D. putrida D. testacea D. recens D·tal/eni Age" Identification index e 2 e 2 e 2 e 2 (key MS ions) 1700 NE 1.4 2.2 1.2 1.8 1.0 1.6 2.0 2.1 Heptadecane (240) SM 1.4 1.6 1.3 1.3 1.4 1.2 2.6 2.4 1900 NE 1.3 2.0 1.0 1.5 1.0 1.6 1.9 2.0 Nonadecane (268) SM 1.4 1.7 1.1 1.1 1.3 1.3 2.3 2.3 2678 NE 7-Heptacosene (378) SM 2.6 1.6 4.2 4.1 21.0 8.5 2866 NE 3.6 6.2 10.0 17.0 7.7 11.0 9.3 9.6 2-Methyloctacosane (365, 393, 408) SM 22.0 21.0 49.0 46.0 33.0 26.0 67.0 66.0 2873 NE 0.6 1.2 0.7 0.8 0.8 1.0 9-Nonacosene (406) SM 6.6 10.0 2.1 2.0 55.0 42.0 2880 NE 3.5 4.0 5.8 8.1 7-Nonacosene (406) SM 1.6 1.5 0.3 0.2 41.0 18.0 3052 NE Hentriacontadiene (432) SM 59.0 35.0 3065-80 NE 36.0 64.0 29.0 56.0 45.0 61.0 73.0 85.0 2-Methyltriacontane (393, 421, 436) + hentriacontenes (434) SM 44.0 52.0 41.0 59.0 69.0 47.0 224.0 156.0 3242-71 NE 11.0 18.0 13.0 24.0 19.0 30.0 23.0 17.0 Tritriacontadienes (460) 2-methyldotriacontane (421, 449, 464), + tritriacontenes (462) SM 29.0 21.0 74.0 103.0 107.0 116.0 100.0 113.0 3445-70 NE 7.6 13.0 14.0 26.0 17.0 23.0 28.0 30.0 Pentatriacontenes (490) + ? SM 28.0 36.0 49.0 61.0 107.0 90.0 6.0 12.0

" NE, newly emerged; SM, sexually mature. phila: ethyl hexanoate is a pheromone compo­ phila, it is likely that the pheromonal effects ob­ nent in D. virilis Sturtevant (Bartelt et al. 1985a) served in our field trials were similarly caused by and is part ofthe male-specific ester blend in five male-specific compounds. other taxa ofthe D. virilis species group (Bartelt Second, it is likely that our chemical data do et al. 1986a). However, its analog, isopropyl hex­ not accurately reflect all the quantitative differ­ anoate, or the alcohol (Z)-1l-octadecen-1-ol have ences among wild flies in the spectrum of not been noted before in any Drosophila ex­ volatile compounds they emit. Because the com­ tracts. pounds differ in molecular weight, they evapo­ Some of these male-specific esters are known rate from the filter paper in the field trials at to originate in the reproductive tract in other different rates. Thus, the relative proportions of species of Drosophila (e.g., Schaner et al. 1987) the different compounds emanating from the fil­ and thus may be most concentrated around areas ter paper change through time (over the course of where flies are mating. Because mycophagous 24 h in our field trials) and probably differ from species ofDrosophila commonly use mushrooms those given off by live flies. Under natural con­ as mating sites (James & Jaenike 1992), such ditions, the relevant variables are rates of pro­ compounds are likely to be liberated there. This duction and volatilization of the different com­ suggests the possibility that, although com­ pounds by live flies. In our field experiments, pounds with pheromonal effects may be pro­ even though fly extracts were applied only once duced only by males, the presence of females every 24 h, we did find significant effects on fly may be required for such effects to be realized. distributions. This suggests that the larger, less Two caveats apply to our results. First, be­ rapidly volatilized components play an impor­ cause we did not compare the attractiveness of tant role in fly responses to resources. In any mushrooms treated with extracts from male ver­ case, the chemical data provide a basis for further sus female flies, we cannot yet conclude that the field experiments involving specific chemical pheromonal effects are caused by male-specific blends. compounds in our species. However, because Despite these caveats, all our observations, in­ known aggregation pheromones are produced cluding both the laboratory analyses of fly ex­ only by mature males in other species of Droso- tracts and the field tests of their effects on fly November 1992 JAENIKE ET AL.: AGGREGATIONS OF MYCOPHAGOUS DROSOPHILA SPP. 703

WHOLE EXTRACT

WHOLE EXTRACT, AFTER No 2 C03

5 15 20 RETENTION TIME (MIN) Fig. 3. Gas chromatograms of extract of mature male D. falleni before (above) and after (below) removal of fatty acids by treatment with Nil:lCOa solution. distributions, support the idea that species­ but these would probably require direct contact specific blends of volatile compounds produced rather than being sensed from a distance because by Drosophila spp. playa role in bringing about of their low volatility. These compounds are aggregations of breeding adults in natural popu­ probably not involved in attracting flies to a site. lations. Thus, we suggest that any mushroom that Intraspecific aggregation is by now well docu­ happens to be found by flies ofa particular spe­ mented in many species of insects that use cies will subsequently be more attractive to patchy, ephemeral resources (Ives 1988a, other flies, particularly those ofthe same species Rosewell et al. 1991). As emphasized by Atkin­ group. son & Shorrocks (1981), such aggregation inten­ The species-specific and mature male-specific sifies intraspecific competition, thus facilitates compounds of higher molecular weight (e.g., the coexistence of ecologically similar species. >30 carbon atoms) may also affect fly behavior, Because coexistence is dependent on species be-

Table 3. Total numbers of flies captured as a function of bait type in the four field trials

No. flies captured Site Dates Bait type D. putrida D. testacea D·falleni D. recens Brighton Town Park 15-18 July D·falleni 32 13 77 1 D. putrida 45 19 63 1 Control 34 9 62 1 Brighton Town Park 12-15 Aug. D·falleni 24 20 84 3 D. putrida 12 14 42 2 Control 12 10 40 3 Powder Mill Park 22-24 July D·falleni 21 13 47 1 D. putrida 21 10 23 0 Control 9 5 11 1 Powder Mill Park 12-15 Aug. D·falleni 85 40 101 4 D..putrida 112 50 90 2 Control 67 45 54 0 704 M"NALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 85, no. 6

Table 4. Analysis of numbers of D. quinaria and D. 1327. testacea group flies captured at baits treated ....ith eitherD. 1989. Aggregation pheromone components in falleni or D. putrida extracts (CATMOD procedure, SAS Drosophila mullen: a chiral ester and an unsatura­ Institute 1985) ted ketone. J. Chern. Ecol. 15: 399-411. Brown, W. L. & E. O. Wilson. 1956. Character dis­ Source of elf p variation :f placement. Syst. Zool. 5: 49-64. Carlson, D. A., C. S. Roan, R. A. Yost & J. Hector. Intercept 1 3.51 0.061 1989. Dimethyl disulfide derivatives oElong chain Trial 3 35.72 0.0001 alkenes, alkadienes, and alkatrienes for gas chroma­ Baits within trials 4 12.54 0.014 tography/mass spectrometry. Anal. Chern. 61: 1564-1571. Elkinton, J. S., C. Schal, T. Ono & R. T. Carde. 1987. Pheromone puff trajectory and upwind flight of ing distributed more or less independently male gypsy moths in a forest. Physiol. Entomol. 12: across breeding sites (Atkinson & Shorrocks 399-406. 1981, Ives 1988b), we suggest that coexistence Grimaldi, D. & J. Jaenike. 1984. Competition in will be facilitated if different species produce natural populations of mycophagous Drosophila. and respond to different sets of volatile phero­ Ecology 65: 1113-1120. Hanski, I. 1987. Colonization of ephemeral habi­ mones, even though the primary function ofsuch tats, pp. 155-185. In A. J. Gray, M. J. Crawley & P. J. pheromones is intraspecific chemical communi­ Edwards [eds.], Colonization, succession, and sta­ cation. If such pheromones playa major role in bility. Blackwell, Oxford. determining the distribution ofinsects across re­ Hoffmann, A. A. & P. A. Parsons. 1986. Inter- and source patches, they might exhibit patterns of intraspecific variation in the response ofDrosophila evolutionary character displacement (Brown & melanogaster and D. simulans to larval cues. Be­ Wilson 1956). hav. Genet. 16: 295-306. Ives, A. R. 1988a. Aggregation and the coexistence of competitors. Ann. Zool. Fenn. 25: 75-88. Acknowledgments 1988b. Covariance, coexistence and the population dynamics of two competitors using a patchy re­ We thank G. Spencer (USDA-ARS National Center source. J. Theor. BioI. 133: 345-361. for Agricultural Utilization Research), T. Phillips (Uni­ 1991. Aggregation and coexistence in a carrion fly versity of Wisconsin), and two anonymous reviewers community. Ecol. Monogr. 61: 75-94. for helpful comments. This research was supported by James, A. C. & J. Jaenike. 1992. Determinants of NSF grant BSR 89-05399 to J. J., including an REU male mating success in wild Drosophila testacea. supplement to support A.F.H.; S. T. and J.S.L. were Anim. Behav. 44:168-170. supported as PEW Summer Research Interns at the Jaenike, J. & A. C. James. 1991. Aggregation and University of Rochester. the coexistence of mycophagous Drosophila. J. Anim. Ecol. 60: 913-928. References Cited Rosewell, J., B. Shorrocks & K. Edwards. 1991. Competition on a divided and ephemeral resource: Atkinson, W. D. & B. Shorrocks. 1981. Competition testing the assumptions. 1. Aggregation. J. Anim. on a divided and ephemeral resource: a simulation Ecol. 59: 977-1001. model. J. Anim. Ecol. 50: 461-471. SAS Institute. 1985. SAS user's guide: statistics. Bartelt, R. J., L. L. Jackson & A. M. Schaner. 1985a. SAS Institute, Cary, NC. Ester components of aggregation pheromone of Schaner, A. M., R. J. Bartelt & L. L. Jackson. 1987. Drosophila virilis (Diptera: Drosophilidae). J. (Z)-ll-Octadecenyl acetate, an aggregation phero­ Chern. Ecol. 11: 1197-1208. mone in Drosophila simulans. J. Chern. Ecol. 13: Bartelt, R. J., A. M. Schaner & L. L. Jackson. 1985b. 1777-1786. cis-Vaccenyl acetate as an aggregation pheromone Schaner, A. M., K. J. Graham & L. L. Jackson. 1989a. in Drosophila melanogaster. J. Chern. Ecol. 11: Aggregation pheromone characterization and com­ 1747-1756. parison in Drosophila ananassae and Drosophila Bartelt, R. J., A. M. Schaner & L. L. Jackson. 1986a. bipectinata. J. Chern. Ecol. 15: 1045-1055. Aggregation pheromones in five taxa of the Droso­ Schaner, A. M., L. L. Jackson, K. J. Graham & R. D. phila vinlis species group. Physiol. Entomol. 11: Leu. 1989b. (Z)-l1-Eicosenyl acetate, an aggre­ 367-376. gation pheromone in Drosophila malerkotliana. J. Bartelt, R. J., M. T. Armold, A. M. Schaner & L. L. Chern. Ecol. 15: 265-273. Jackson. 1986b. Comparative analysis of cutic­ Throckmorton, L. H. 1975. The phylogeny, ecology, ular hydrocarbons in the Drosophila virilis and geography ofDrosophila, pp. 421-469. In R. C. species group. Compo Biochem. Physiol. B. Compo King [ed.], Handbook of genetics, vol. 3. Plenum, Biochem. 83: 731-742. New York. Bartelt, R. J., A. M. Schaner & L. L. Jackson. 1988. Aggregation pheromones in Drosophila borealis Received for publication 30 December 1991; ac­ and Drosophila littoralis. J. Chern. Ecol. 14: 1319- cepted 6 July 1992.