690 9 ECOLOGY AND POPULATION BIOLOGY Aggregations in Mycophagous Drosophila (Diptera: Drosophilidae): 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 mushrooms 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 flies 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 insect 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 insects 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-fly 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 mushroom, tifications; some low-intensity ions were not ob­ Agaricus 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