Journal of Chemical Ecology, Vol. 26, No. 5, 2000 AGGRESSIVE CHEMICAL MIMICRY BY THE BOLAS SPIDER Mastophora hutchinsoni: IDENTIFICATION AND QUANTIFICATION OF A MAJOR PREY’S SEX PHEROMONE COMPONENTS IN THE SPIDER’S VOLATILE EMISSIONS CESAR´ GEMENO, KENNETH V. YEARGAN, and KENNETH F. HAYNES* University of Kentucky Department of Entomology Lexington, Kentucky 40546-0091 (Received May 13, 1999; accepted January 11, 2000) Abstract—The adult female bolas spider Mastophora hutchinsoni feeds exclusively on attracted males of a few moth species. This exclusivity and the behavior of the approaching moths suggest that the spider aggressively mimics the sex pheromones of its prey species. Males of the bristly cutworm, Lacinipolia renigera, are a major prey of this spider, accounting for about two thirds of the biomass of prey consumed. Female bristly cutworms produce a pheromone blend consisting of (Z)-9-tetradecenyl acetate (Z9–14 : Ac) and (Z,E)-9,12-tetradecenyl acetate (ZE-9,12–14 : Ac). To determine if M. hutchinsoni females mimic the sex pheromone components and blend ratio of L. renigera, we collected volatiles from hunting adult female spiders and analyzed them with gas chromatography–electroantennographic detection (GC-EAD) and gas chromatography–mass spectrometry (GC-MS). GC-EAD analysis of volatile collections, using a male bristly cutworm antenna as the detector and two capillary columns of different polarities, revealed the presence of peaks with retention times (Rts) identical to Z9–14 : Ac and ZE-9,12–14 : Ac. The mass spectrum of a peak with Rt of Z9–14 : Ac was identical to the mass spectrum of the synthetic equivalent. There was an insufficient quantity of the compound with Rt of ZE-9,12–14 : Ac to get a full spectrum, but selective detection of ions at m/ z 61 and 192 at the correct Rt supported the identification. On average, the blend collected from spiders contained 54.8 ± 20.8 (SE) pg/ min of Z9–14 : Ac and 2.5 ± 1.7 (SE) pg/ min of ZE-9,12–14 : Ac. The latter, on average, comprised 2.6 ± 0.7% of the total, which is similar to the blend ratio emitted by bristly cutworm females. Our *To whom correspondence should be addressed. 1235 0098-0331/ 00/ 0500-1235$18.00/ 0 2000 Plenum Publishing Corporation 1236 GEMENO, YEARGAN, AND HAYNES results indicate that the adult female M. hutchinsoni produces an allomone blend that mimics not only the composition, but also the blend ratio, of the sex pheromone of a major prey species. Key Words—Insect pheromones, pheromone emission, aggressive chemical mimicry, predation, allomone, Araneae, Araneidae, Lepidoptera, Noctuidae. INTRODUCTION Adult female bolas spiders prey exclusively on males of a restricted number of moth species (Stowe et al., 1987; Yeargan, 1994). The spider’s bolas is a highly specialized device consisting of a ball of sticky material suspended from a short thread. The spider swings the bolas at an approaching male moth, and the liquid component of the bolas penetrates through the scales of the moth to the under- lying cuticle, allowing the spider to capture its victim (Eberhard, 1980; Year- gan, 1994). Eberhard (1977) provided the first convincing evidence of aggressive chemical mimicry in a bolas spider based on his observations and field exper- iments with Mastophora dizzydeani Eberhard in South America. Stowe et al. (1987) provided chemical evidence for aggressive mimicry by identifying com- pounds in the volatile emissions from Mastophora cornigera Hentz that corre- sponded with some of the pheromone components of its moth prey species. The adult female bolas spider Mastophora hutchinsoni Gertsh preys on four moth species in Kentucky, of which the bristly cutworm, Lacinipo- lia renigera (Stephens), comprises 40% of the total number of prey cap- tured and approximately two thirds of the biomass consumed (Yeargan, 1988). The bristly cutworm sex pheromone blend consists of (Z)-9-tetradecenyl acetate (Z9–14 : Ac) and 3.8% (Z,E)-9,12-tetradecenyl acetate (ZE-9,12–14 : Ac) (Haynes, 1990). Because Z9–14 : Ac alone failed to attract male bristly cut- worm moths, we suspected that the spider must mimic both components in an appropriate ratio in order to be an effective predator. We tested this hy- pothesis by analyzing volatile compounds emitted by female M. hutchinsoni with gas chromatography–electroantennographic (GC-EAD) detection and gas chromatography–mass spectrometry (GC-MS). METHODS AND MATERIALS Study Organisms. Egg sacs of M. hutchinsoni were collected from trees during the winter in central Kentucky and were relocated to tree lines on farms owned by the University of Kentucky. During the late summer and early fall, adult female spiders were collected from these farms and placed on 1.5-m-tall apple trees housed in an outdoor walk-in cage (3.7 × 3.7 × 2.1 m maximum BOLAS SPIDER MIMICRY 1237 height) made of fine-mesh nylon screen. When spiders were hunting, they were hand-fed at least once a week with adult male cabbage looper moths, Trichoplu- sia ni (Hubner),¨ which is not a prey species. Lacinipolia renigera and T. ni were reared in the laboratory on a pinto bean diet following the procedures of Haynes (1990) and Shorey and Hale (1965), respectively. Male pupae were placed in environmental chambers under a 13L:11D photoregime, with a L : D temperature cycle of 258C:218C. Volatile Collections. During the period of mid-August through October, 1998, volatile compounds emitted by spiders were collected (outdoors) between 8 and 10 PM, corresponding to the seasonal and daily periods when male L. renigera are captured by female M. hutchinsoni (Yeargan, 1988). The collector consisted of a glass funnel, 60 mm long × 17.5 mm cup ID × 3.5 mm stem ID (Supelco, Bellefonte, Pennsylvania), packed with 20 mg of activated charcoal, 16–30 mesh (SGE, Inc., Austin, Texas). A vacuum pump (Gast model ROA- P131-AA, Bent Harbor, Michigan) was used to create a flow through the col- lector of 6 liters/ min. The collector was placed 2 cm downwind of a hunting spider and was held in place with a modified microphone stand (MCM Elec- tronics, Centerville, Ohio). If the wind changed direction during a collection, the collector was repositioned so that it again was directly downwind of the spider. Collections continued until the spider stopped hunting. Collectors were rinsed with 2 ml of CH2Cl2. Depending on the duration of the collection, all or part of this volume was subsequently reduced to 2 ml for analysis. Gas Chromatography–Electroantennographic Detection. We used GC- EAD to identify and quantify the volatile compounds emitted by the spiders. The system was similar to that described by Struble and Arn (1984) and Haynes et al. (1996). A Hewlett-Packard 5890 Series II GC was equipped with either a DB-5 or a DB-Wax column (both columns 30 m × 0.25 mm ID, J & W Sci- entific, Folsom, California). The effluent from the column was split at a 1 : 1 ratio between a flame ionization detector (FID) and an EAD detector. The oven temperature was initially held at 808C for 2 min, and then increased to 2308C at a rate of 208C/ min. Just prior to the split, a nitrogen makeup gas flow rate (70 ml/ min) was introduced with a VSIS-5 T connector (Scientific Glass Engineer- ing, Austin, Texas). The effluent flowed from the T connector through a 5-cm section of deactivated column (0.53 mm ID), which was inserted into a deacti- vated glass Y connector (Restek, Bellefonte, Pennsylvania). One branch of the Y connected to the FID; the other branch connected to a modified glass condenser where the antennal preparation was located. Humidified air flowed through the glass condenser at 2 liters/ min delivering the GC effluent to the antennal prepa- ration. The condensor was cooled with ice water to increase the longevity of the antennal preparation. The electrophysiological preparation involved placing the proximal end of a male L. renigera antenna into a pool of an insect saline solution (Pringle, 1938). 1238 GEMENO, YEARGAN, AND HAYNES Several terminal segments of the antenna were removed, and the distal end of the antenna was placed into a separate pool of saline. Silver–silver chloride elec- trodes were placed into each drop of saline and connected through a Grass P16 high-impedance probe to a Grass P16 Amplifier (Grass Medical Instruments, Quincy, Massachusetts). The passive high-pass filter described by Struble and Arn (1984) controlled baseline drift. The amplified EAD and FID signals were sent to a DI-420 Signal Conditioning Module (Dataq Instruments Inc., Akron, Ohio). Both signals were recorded and analyzed by Dataq software. All synthetic compounds were obtained from IPO-DLO, Wageningen, The Netherlands. Isomeric and overall purity was greater than 99% (Simon Voerman, Research Chemist, IPO-DLO). After potential moth pheromone components had eluted from the GC, two consecutive 5-cm3 puffs of air containing Z9–14 : Ac were introduced into the air flow over the antenna through a syringe. These standard puffs were generated from a glass pipet cartridge containing 100 ng of Z9–14 : Ac loaded onto a 1.5- cm2 piece of fluted filter paper. The absolute amplitude of an EAG response reflects the quantity of compound present, but also is affected by the condition of the antenna and variation among males. Therefore, to estimate the quantity of pheromone present we corrected for the responsiveness of the antenna by dividing the amplitude of the GC peak by the amplitude of the standard puff of Z9–14 : Ac. This standardization yields a unitless ratio that is very consistent from injection to injection for a given dose. This ratio was determined for a wide range of doses (0.1 pg–10 ng of both Z9–14 : Ac or ZE-9,12–14 : Ac, N c 3 per dose), and a best fit regression equation between dose and ratio was determined with Sigma Plot 4.0 for Windows (SPSS, Chicago, Illinois).