Aggressive Chemical Mimicry by the Bolas Spider Mastophora Hutchinsoni

Home , Bolas spider, Lacinipolia renigera

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 compounds 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 captured 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 cutworm 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 collector 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 deactivated 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 preparation. 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). The GC-MS analysis was performed on a Hewlett-Packard 5890 Series II Plus GC with a Hewlett-Packard 5972 Mass Selective Detector. The same type of DB-Wax column that was used for GC-EAD analysis was installed in this GC and operated under the same conditions, with the exception that the column temperature was raised at 108C/ min. Sixty percent of a 62-min collection was analyzed in the scan mode (50–350 m/ z). A small fraction of the sample had previously been analyzed by GC-EAD to ensure that there was sufficient quantity of the putative Z9–14 : Ac peak to yield a spectrum. To gain added sensitivity necessary to detect the minor component, 70% of a pooled sample (138 min of collections from six spiders) was analyzed in the selected ion mode, focusing on ions characteristic of the acetate groups, ZE-9,12–14 : Ac and Z9–14 : Ac (m/ z 61, 192, and 194, respectively).

RESULTS AND DISCUSSION

Two EAG peaks with the same retention times as synthetic Z9–14 : Ac and ZE-9,12–14 : Ac were observed using DB-Wax and DB-5 capillary columns BOLAS SPIDER MIMICRY 1239

FIG. 1. GC-EAD responses of male Lacinipolia renigera to 0.05 ng of synthetic Z9–14 : Ac and ZE-9,12–14 : Ac (top) and to volatile samples collected from hunting female Mastophora hutchinsoni (bottom). The collections analyzed on DB-Wax and DB- 5 were 19.0 min and 12.4 min in duration, respectively. FID traces are not shown.

(Figure 1). Kovats indices (Jennings, 1980) for these two peaks were 2142 and 2222, respectively, with a DB-Wax column, and 1799 and 1811, respectively, with a DB-5 column. Electroantennographic detection of compounds had a twofold advantage in this system. First, the emission rate from many spiders was substantially below the lower limits of detection of our flame ionization system, and thus the greater sensitivity of the EAD system was essential. Sec- ond, because the collection system was open and collections were performed outdoors, we collected many additional compounds from the environment, and thus the specificity of using male L. renigera antennae as detectors gave added confidence in the identity of the compounds. Regression of the log-transformed quantity of Z9–14 : Ac (or ZE- 9,12–14 : Ac) versus the log-transformed standardized GC-EAD response (GC- EAD peak amplitude divided by the amplitude of a standard puff) was highly significant (P < 0.001 for both compounds) (Sokal and Rohlf, 1995). The regression equations for Z9–14 : Ac and ZE-9,12–14 : Ac explained 88% and 91%, respectively, of the variance of responses over a range of injected concentrations from 0.1 pg to 10 ng (Figure 2). Using this calibration curve, we calculated the emission rate of both compounds from volatile collections from hunting female M. hutchinsoni (Figure 3). Z9–14 : Ac was collected from hunting spiders at a 1240 GEMENO, YEARGAN, AND HAYNES

FIG. 2. Calibration curves relating the quantities (Q) of Z9–14 : Ac (filled circles) and ZE- 9,12–14 : Ac (open circles) to the ratio (R) of the GC-EAD peak amplitude to a standard puff amplitude. The regression equations explained 88 (P < 0.001) and 91% (P < 0.001) of the variation in the quantities of Z9–14 : Ac and ZE-9,12–14 : Ac, respectively. rate of 54.8 ± 20.8 pg/ min (mean ± SE, N c 12), and ZE-9,12–14 : Ac at a rate of 2.5 ± 1.7 pg/ min (mean ± SE, N c 12). This open collection system samples a portion of the volatile compounds that are actually emitted by the spider, and its efficiency is related to the relationship between the ambient wind speed, the accuracy of estimated wind direction, and the flow rate through the collector (6 liters/ min). With a closed collection system that had 90–100% collection efficiency (Baker et al., 1981), Z9–14 : Ac was previously found to be emitted from forcibly extruded sex pheromone glands of female L. renigeria at a rate of 780 pg/ min (Haynes, 1990). The mean percentage of ZE-9,12–14 : Ac in the blend emitted by the spider was 2.6 ± 0.7% (mean ± SE, N c 12), which is similar to the mean percentage observed in L. renigera (3.8%) (Haynes, 1990). The mass spectrum of the putative Z9–14 : Ac peak matched that of authentic Z9–14 : Ac, with a correspondence in the relative quantities of abundant ions at m/ z 55, 67, 81, 82, 96, 110, and 123, as well as a good match between the ion characteristic of acetates (m/ z 61), and the ion most characteristic (M+- CH3COOH) of tetradecenyl acetate (m/ z 194). The ratio of ions at m/ z 55 to m/ z 54 was 1.97 for authentic Z9–14 : Ac and 2.07 for the corresponding compound in the volatile collection, suggesting that the double-bond position was BOLAS SPIDER MIMICRY 1241

FIG. 3. A GC-EAD trace for a 21.5-min volatile collection from a female bolas spider, Mastophora hutchinsoni. Quantification of Z9–14 : Ac and ZE-9,12–14 : Ac was done by using the regression equation (see text and Figure 2) relating quantity to the ratio of GC- EAD peak amplitude to standard puff amplitude. These standard peaks were generated by two consecutive 5-cm3 puffs of air through a glass pipet cartridge containing 100 ng of Z9–14 : Ac loaded onto a 1.5-cm2 piece of fluted filter paper. at the 9 position (Leonhardt et al., 1983). Z8–14 : Ac and Z10–14 : Ac yielded ratios of m/ z 55 to m/ z 54 of 1.66 and 2.41, respectively. The molecular ions for authentic Z9–14 : Ac or the corresponding peak in the volatile collection were not detected at these low quantities. ZE-9,12–14 : Ac was not detected in the full-spectrum scan mode. However, with another sample, using the more sensi- tive selected ion mode, we observed peaks at the retention times of synthetic Z9–14 : Ac (m/ z 61 and 194) and ZE-9,12–14 : Ac (m/ z 61 and 192). These results support the evidence that the peaks observed in the EAD analysis corre- spond with the pheromone components of L. renigera. Electrophysiological and chemical evidence support the presence of Z9–14 : Ac and ZE-9,12–14 : Ac in volatile collections from hunting female M. hutchinsoni. One previous study has reported an overlap between the compounds used by a bolas spider, M. cornigera, and the pheromone components of some of its lepidopteran prey (Stowe et al., 1987). In addition to identifying a similar overlap of components, we found that the blend ratio of Z9–14 : Ac and ZE-9,12–14 : Ac emitted by M. hutchinsoni is very similar to that produced by female L. renigera. In moths, the blend ratio of pheromone components is often critical to optimal attraction of mates (Carde´ and Charlton, 1984), and likewise the spider’s predatory success would be dependent on its effectiveness in mim- icking the blend ratio of pheromone components. The identification of these two allomonal (sensu Whitaker and Feeny, 1971) 1242 GEMENO, YEARGAN, AND HAYNES components M. hutchinsoni is only part of the story of aggressive chemical mimicry in this species. The most abundant prey species of late-instar female M. hutchinsoni in Kentucky during late July and the first two thirds of August, when males of L. renigera are not available, is the smoky tetanolita moth, Tetanolita mynesalis (Walker). This moth uses a pheromone consisting of a 2 : 1 blend of (3Z,9Z)-(6S,7R)-epoxy-heneicosadiene and (3Z,6Z,9Z)-heneicosatriene (Haynes et al., 1996). The pheromone blend for the bronzed cutworm, Nephelodes mini- mans Guennee,´ a minor prey species, consists of (Z)-11-hexadecenal and (Z)-11- hexadecenyl acetate (Zhu and Haynes, unpublished data). Although a pheromone for the fourth prey species (also minor), Parapediasia teterrella (Zincken), has not been identified, a very effective sex attractant consists of a 20 : 1 blend of (Z)-11-hexadecenal and (Z)-9-hexadecenal (Clark and Haynes, 1990). The spider’s optimization of a blend for one species may have the asso- ciated cost of diminished effectiveness in attracting other species. For exam- ple, the pheromone blend of L. renigera interferes with the attraction of male T. mynesalis (Haynes and Yeargan, unpublished data). Alternatively, the spider may have some chemical and/ or behavioral plasticity in the synthesis and release of various allomonal components, such that this behavioral antagonism is reduced. Aggressive chemical mimicry in M. hutchinsoni is even more complex when one considers the spider’s entire life cycle. Juvenile spiders of both sexes and adult males attract male psychodid flies (moth flies) (Yeargan and Quate, 1996, 1997), but pheromones have not been identified for any species of moth fly. From the current study it is clear that during the season and at the time of night when L. renigera is active, adult female M. hutchinsoni produce a blend of Z9–14 : Ac and ZE-9,12–14 : Ac that is very effective in attracting male L. renigera. This is an important part of pheromone mimicry by this spider because L. renigera is the major source of nutrients for the spider during the period when females produce eggs. However, it is clear that the complete story of aggressive chemical mimicry in this species will be complex, and will require studies of other species of prey and other developmental stages of the spider.

Acknowledgments—We thank B. Chastain, K. Johnson, and S. Mayes for their technical assis- tance. Drs. L. Rieske-Kinney and M. Sharkey reviewed an early draft of this manuscript. This material is based upon work supported by the National Science Foundation under grant IBN-97-22828. The GC-MSD system was purchased in part by funds from the University of Kentucky major equip- ment program (MRES94-01-Haynes). This investigation (paper no. 99-08-54) was conducted in con- nection with two projects of the Kentucky Agricultural Experiment Station.

REFERENCES

BAKER, T. C., GASTON, L. K., POPE, M. M., KUENEN, L. P. S., and VETTER, R. S. 1981. A high- efﬁciency collection device for quantifying sex pheromone volatilized from female glands and synthetic sources. J. Chem. Ecol. 7:961–968. BOLAS SPIDER MIMICRY 1243

CARDE´ , R. T., and CHARLTON, R. E. 1984. Olfactory sexual communication in Lepidoptera: Strategy, sensitivity and selectivity, pp. 241–265, in T. Lewis (ed.). Insect Communication. Academic Press, London. CLARK, J. D., and HAYNES, K. F. 1990. Sex attractant for the bluegrass webworm (Lepidoptera: Pyralidae). J. Econ. Entomol. 83:856–859. EBERHARD, W. G. 1977. Aggressive chemical mimicry by a bolas spider. Science 198:1173–1175. EBERHARD, W. G. 1980. The natural history and behavior of the bolas spider Mastophora dizzydeani sp. n. (Araneidae). Psyche 87:143–169. HAYNES, K. F. 1990. Identification of sex pheromone of bristly cutworm, Lacinipolia renigera (Stephens). J. Chem. Ecol. 16:2615–2621. HAYNES, K. F., YEARGAN, K. V., MILLAR, J. G., and CHASTAIN, B. B. 1996. Identification of sex pheromone of Tetanolita mynesalis (Lepidoptera: Noctuidae), a prey species for bolas spider, Mastophora hutchinsoni. J. Chem. Ecol. 22:75–89. JENNINGS, W. 1980. Gas Chromatography with Glass Capillary Columns, 2nd ed. Academic Press, New York. LEONHARDT, B. A., DEVILBISS, E. D., and KLUN, J. A. 1983. Gas chromatographic mass spectro- metric indication of double bond position in monounsaturated primary acetates and alcohols without derivatization. Org. Mass Spectrom. 18:9–11. PRINGLE, J. W. S. 1938. Proprioreception in insects. I. A new type of mechanical receptor from the palps of the cockroach. J. Exp. Biol. 15:101–113. SHOREY, H. H., and HALE, R. L. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. J. Econ. Entomol. 58:522–524. SOKAL, R. R., and ROHLF, F. J. 1995. Biometry, 3rd ed. W. H. Freeman, New York. STOWE, M. K., TUMLINSON, J. H., and HEATH, R. R. 1987. Chemical mimicry: Bolas spiders emit components of moth prey species sex pheromones. Science 236:964–967. STRUBLE, D. L., and ARN, H. 1984. Combined gas chromatography and electroantennogram record- ing of insect olfactory responses, pp. 161–178, in H. E. Hummel and T. A. Miller (eds.). Tech- niques in Pheromone Research. Springer-Verlag, New York. WHITAKER, R. H., and FEENY, P. P. 1971. Allelochemics: Chemical Interactions between species. Science 171:257–270. YEARGAN, K. V. 1988. Ecology of a bolas spider, Mastophora hutchinsoni: Phenology, hunting tactics, and evidence for aggressive chemical mimicry. Oecologia 74:524–530. YEARGAN, K. V. 1994. Biology of bolas spiders. Annu. Rev. Entomol. 39:81–99. YEARGAN, K. V., and QUATE, L. W. 1996. Juvenile bolas spiders attract psychodid flies. Oecologia 106:266–271. YEARGAN, K. V., and QUATE, L. W. 1997. Adult male bolas spiders retain juvenile hunting tactics. Oecologia 112:572–576.