Journal ofChemical Ecology. Vol. 23. No.8. 1997 7821

CHEMISTRY AND DEFENSIVE EFFICACY OF SECRETION OF BURROWING BUG ( cinctus cinctus)

BRYAN S. KRALL,I.* BRUCE W. ZILKOWSKI,2 SCOTT L. KIGHT,3 ROBERT J. BARTELT,2 and DOUGLAS W. WHITMAN!

'4120 Biological Sciences. J//inois State University Normal. J//inois 61790 1USDA. ARS Narional Center for Agricultural Utilization Research Bioactive Agents Research Unit 1815 N. University. Peoria. J//inois 61604 3Depanment ofBiology and Center for the Integrative Study ofAnimal Behavior Indiana University Bloomington. Indiana 47405

(Received September 16, 1996; accepted April 2, 1997)

Abstract-Adult cinctus emit a volatile secretion from their metathoracic scent glands when tactually stimulated. We identified the volatile components by gas chromatography, high-performance liquid chromatogra­ phy. and mass spectrometry. The secretion of both sexes contained (lR)-(+)­ a-pinene. (lS)-( - )-,B-pinene, ,B-myrcene, (R)-( + )-limonene, and a-terpino­ lene. Two additional compounds were found in only female secretions: (E)-2-hexenyl acetate and (E)-2-octenal. We also tested the defensive capa­ bility of this by offering it to various predators. Anoles, starlings, and a killdeer rejected S. c. cinctus after an initial sampling. These findings sug­ gest that the secretion plays a defensive role.

Key Words-Terpenes, chemical defense, , , Sehirus cinctus cinctus. (IR)-( + loa-pinene, (IS)-( - )-,B-pinene. ,B-myrcene, (R)-( +)­ limonene. a-terpinolene. (E)-2-hexenyl acetate, (E)-2-octenal.

INTRODUCTION

Hemipterans are well known for their use of repellent chemicals to defend themselves against predators (Staddon, 1979; Blum, 1981; Aldrich, 1988).

*To whom correspondence should be addressed.

1951

0098.()33119710800-1951SI2.5010 @ 1997 Plenum Publishing Corporation 1952 KRALL ET AL.

However, not much is known about the defensive chemistry of cydnids, a cos­ mopolitan family closely related to the Pentatomidae (Miller, 1971). Even less is known about chemical defense in the cydnid subfamily . In the United States, the Sehirinae are represented by one genus containing one species and three subspecies. The most common subspecies, Sehirus cinctus cinctus (Palisot de Beauvois), ranges from Massachusetts to Florida and as far west and south as Iowa, New Mexico, and Mexico (Froeschner, 1960). Cydnids in general, and S. c. cinctus in particular, are unique in that they practice brood care in underground chambers (Sites and McPherson, 1982). Adult S. c. cinctus overwinter and emerge in late March to April to feed in aggregations on several species of mint. By the end of April, adults mate and move to the ground (Sites and McPherson, 1982), where the females find or 3 dig a small (ca. 0.5 cm ) chamber approximately 2 cm below the soil surface and lay a batch of 120-150 eggs. Females remain with the eggs and early-instar larvae for several weeks, guarding them against ants and other predators and feeding them by carrying mint seeds from the surface'to the chamber (Southwood and Hine, 1950; McDonald, 1968). The nymphs leave the brood chamber when they reach the third instar and travel to the surface to feed on mint foliage (Sites and McPherson, 1982). They pass through five instars before reaching adulthood and can be found between early May and mid-August, while adults can be found from early Spring to late Summer. S. c. cinctus nymphs are aposematically colored. They are black with reddish orange markings on the head, thorax, and abdomen. Adults may also be aposematic when viewed against green vegetation. They are small (ca. 5 mm long), black with a thin cream-colored stripe along the lateral margins of each wing beginning at the head and extending down to the anterior of the wings. When stimulated by lightly pinching the legs or antennae, the adults emit a volatile secretion. This suggests that the secretion functions in chemical defense against predators.

METHODS AND MATERlALS

Insect Collection and Care Adult S. c. cinctus were collected on wild mints near Bloomington, Indiana, in early April 1995. The bugs were then kept in a cold chamber for three months at 7°C under a lOL: 14D photoperiod to preserve them until needed. After retrieval from the cold chamber, individuals were maintained according to Sites and McPherson (1982) with the following modifications: no plastic wrap was placed over the Petri dish, as this induced harmfully high humidities. Instead, a l-cm2 screened ventilation hole was placed in the center of each plastic Petri BURROWING BUG SECRETIONS 1953 dish lid. Vial caps containing dried fruits ofPrunella vulgaris (Labiatae) allowed the bugs to feed ad libitum.

Chemical Analysis Chemicals. (1S)-( - )-a-Pinene (98%), dl-a-pinene (99%), (1R)-( +)-{3­ pinene (98 %), (1S)-( - )-{3-pinene (99 %), (R)-( +)-limonene (97 %), (S)-( -)­ limonene (96%), a-terpinolene (99%), and (E)-2-octenal (94%) were purchased from Aldrich Chemical Co., Milwaukee, Wisconsin. J3-Myrcene (90%) and (E)-2-hexenyl acetate (97 %) were obtained from Sigma Chemical Co., St. Louis, Missouri. Volatile Collection and Analysis. The sex of ca. 25 S. c. cinctus adults was detennined using a microscope, and the insects were placed individually into Hewlett-Packard autosampler vials (glass, 12 mm x 32 rom, with an II-rom Teflon-lined aluminum crimp cap). A 500-Jotl gas-tight syringe was used to draw 200 Jotl of headspace volatiles out of each vial. Volatiles were then immediately injected into a GC for analysis (Hewlett-Packard 5890, equipped with splitless injector, and flame ionization detector). The DB-l capillary column (J &W Scientific, Folsom, California) was 15 m x 0.25 rom and had a 1.0-Jotm film thickness. The temperature program was 50-150°C at lOoC/min. Following analysis of the first gas sample, the cap was removed from the vial, and the insect was agitated by either grabbing a leg with forceps or holding the individual against the sides or bottom of the vial until the characteristic odor was obvious to the investigator. (In some cases merely attempting to grab an insect without actually restraining it was enough to elicit release of the secretion). At the first hint of chemical release, a new cap was tightly crimped on to the vial and a second headspace volatile sample (200 Jot!) was removed and injected into the GC. A second method of volatile collection was used for ca. 50 unsexed dead adult S. c. cinctus. Dead adults (deceased adults stored at -SoC within 24 hr of death and kept up to one month before extraction) were covered with 5 rnl of hexane and then crushed with a glass rod. The sample was filtered, reduced in volume under Nz, and then fractionated isocratically with high-perfonnance liquid chromatography (HPLC) using a Waters Associates 6000 pump and a Waters Associates R401 refractometer detector. A silica column (Lichrosorb Si60, 25 cm x 0.46 cm, 5-Jotm particle size) was eluted with hexane (flow 1 rnI/min) and effluent was collected as consecutive l-rnl fractions. These frac­ tions were analyzed by GC. The fractions provided a larger amount of material than from single headspace collections, making it possible to obtain mass spectra for the insect-derived compounds and to determine chirality for some com­ pounds. Mass spectra were obtained on a Hewlett-Packard 5970 MSD instru­ ment, with sample introduction through a DB-l capillary column (15 m x 0.25 1954 KRALLETAL. mm ID with 1.0-lLm film thickness, temperature program 50-250°C increased at lOoC/min). Chirality was detennined on a chiral GC column by comparison to authentic standards. A 30-m x 0.25-mm-ID CDX-B fused silica capillary column (J & W Scientific) with a 0.25-lLm film thickness was used (temperature program 40-75°C at lOoC/min, then holding at 75°C for 30 min). Seed Extraction. Prunella vulgaris seeds were cleaned of debris and 0.5 g was placed into a glass vial with 2 ml of hexane; the seeds were crushed with a glass rod. The extract was filtered and analyzed by GC (Hewlett-Packard 5890, 15-m x 0.25-mm-ID DB-I capillary column with 0.25-lLm film thickness, temperature program 50-250°C increased at lOoC/min).

Predator Trials Anolis carolensis carolensis. Six adult anole lizards, obtained from a local pet shop, were maintained for two weeks at 2rC with surplus food (various ) and water. The anoles were isolated and starved for two days prior to testing. Each anole was tested individually in its home container by gently introducing a series of adult S. c. cinctus. Houseflies (Musca domestica) and small, second- to fourth-instar Acheta domesticus crickets served as control prey. To begin each test, an anole was initially offered a control insect to verify hunger. Next, S. c. cinctus adults were sequentially offered until the anole refused to attack them. This two-step process was repeated until the anole con­ tinually rejected S. c. cinctus, yet continually ate controls. This process was repeated two or three days later. Onychomys leucogaster. Two laboratory-reared northern grasshopper mice were obtained from the Care Facility at illinois State University. Mice were starved for 4 hr prior to testing and were tested in their home containers. Third- and fourth-instar A. domesticus crickets were used as controls. Each mouse was offered a control cricket followed by five S. c. cinctus in sequence. Charadrius vociferus. A l-week-old killdeer was reared in the lab on a diet of arthropodS, earthwonns, snails, fish and fruit. At about 4 weeks of age, when the bird could feed by itself and discriminate among prey (i.e., began to prefer some prey items over others), it was tested in the following method: The bird was given a control third-instar A. domesticus cricket to demonstrate hunger. Then S. c. cinctus individuals were sequentially offered until the bird no longer consumed the bugs. When this occurred, the bird was then offered another control cricket to verify that the bird was still hungry. If the control was eaten, then another bug was offered. The experiment ended when the bird continued to eat the controls, but repeatedly rejected the bugs. Sturnus vulgaris. A 3-week-old European starling was reared in captivity on a diet of arthropods, fruit, and cat food. When the bird showed strong food BURROWING BUG SECRETIONS 1955 discrimination patterns (Le., began to prefer some foods over others), it was tested. The feeding trials were conducted in the same way as for S. vociferus.

Source ofSecretion Three adult males and three adult females were chilled to lOoC and then glued ventral side up onto cardstock. They were then warmed ·to room temper­ ature, viewed under a dissecting microscope, and gently pinched with tweezers 2 to elicit expulsion of the secretion. Slivers of filter paper (3 mm ) were then placed on the metathoracic gland opening, beak, or anus to absorb any ejected fluids.

RESULTS

Chemical Analysis Headspace Analysis by Gc. No volatiles were detected from nonagitated adult male or adult female S. c. cinctus. However, both male and female S. c. cinctus produced a blend ofat least seven compounds when agitated (Figure 1). Five of the compounds were abundant enough for good-quality mass spectra to be obtained and were tentatively identified by matching to library spectra. These identifications were confirmed when authentic standards gave identical mass spectra and GC retention times (Table I, Figure 2). Of the five terpenes identified, a-pinene, ,B-pinene, and limonene have asymmetric centers. The CDX-B capillary column separated all three pairs of enantiomers, based on the commercial standards: (1S)-( -)- and (1R)-( +)-a­ pinene eluted at 13.5 and 13.8 min, respectively; retentions of (IS)-(-)- and (1R)-( +)-,B-pinene were 18.0 and 18.2 min, respectively; and (S)-( -)- and (R)-( +)-limonene eluted at 21.4 and 21.7 min, respectively. The insect-derived samples had GC peaks for these terpenes only at 13.8, 18.0, and 21.7 min. Therefore, (1R)-( +)-a-pinene, (1S)-( - )-,B-pinene, and (R)-( +)-limonene were the only detectable enantiomers in the S. c. cinctus volatiles. Subsequently, coinjection with selected commercial terpene enantiomers confirmed that GC retention times were stable and that chirality assignments were correct. Two additional peaks (Figure IB) were detected in six of 12 female col­ lections but were never seen in any of the nine male collections. Averaged over the females actually showing these peaks, the two compounds represented 21 % of the female secretion. The compounds were tentatively identified as (£)-2­ hexenyl acetate and (£)-2-octenal from the mass spectra (Table 1), and these identifications were then confirmed when authentic standards produced identical spectra and GC retentions. Ignoring the (£)-2-octenal and (£)-2-hexenyl acetate present in some female 1956 KRALL ET AL.

1 3 1 3 9 A B

4 / 4 /

6 8 6

\ 5 7

4 6 8 4 6 8 Time (min) Time (min) FIG. I. Gas chromatograms of volatile emissions from adult (A) male and (B) female S. c. cinCIUS. Peak numbers correspond to those in Table I and Figure 2. samples, the relative compositions of volatile blends were not significantly dif­ ferent (two-sample t test) between males and females. Pooled over both sexes (N = 10), the mean percentages for terpenes were: (1R)-( +)-a-pinene, 46.6% (SE ± 0.46); (1S)-( - H3-pinene, 43.7% (SE ± 0.49); i3-myrcene, 6.2% (SE ± 0.16); (R)-( +)-limonene, 3.2% (SE ± 0.07); a-terpinolene, 0.4% (SE ± 0.02). Seed Extraction. Analysis of P. vulgaris seeds yielded no trace of the identified compounds by GC analysis. BURROWING BUG SECRETIONS 1957

TABLE 1. CHEMICALS IDENTIFIED IN S. C. cinctus SECRETIONS

GC peak Identification" Major MS ionsb and (percentages)

(1R)-( +i-a-pinene 136 (M+. 7), 93 (100), 91 (48), 79 (28), 77 (39), 41 (26) 2 (unidentified) not available 3 (1SH - )-I3-pinene 136 (M+, 8), 93 (100), 91 (31),77 (31), 69 (35),41 (60) 4 l3-myrcene 136 (M+, 2), 93 (63), 91 (16), 79 (13), 69 (56), 41 (100) 5 (unidentified) not available 6 (R)-( +)-limonene 136 (M+, 17), 93 (83), 77 (33), 68 (100), 67 (82), 41 (40) 7 Terpinolene 136 (M+, 57), 121 (76), 93 (100), 91 (55), 79 (52) 8e (E)-2-hexenyl acetate 142 (M+, 0.4), 100 (12), 82 (22), 67 (36), 57 (13), 55 (16),43 (100), 41 (23) (E)-2-octenal 126 (M+, 0.1),83 (53),82 (27), 58 (14), 57 (46), 55 (84), 43 (16), 42 (36), 41 (100)

"Chirality determined on chiral GC column; set text. bMajor MS ions (m/z). in order of decreasing abundance. cCompounds detected only from some female samples; see text.

Predator Trials

Anolis carolensis carolensis vs. Sehirus cinctu~ cinctus. Of the six anoles tested, two were eliminated because they did not attack the initial control insect. The remaining four anoles developed strong predator avoidance towards S. c. cinctus (Table 2). During the feeding trials, all four anoles behaved in a similar fashion. Each anole quickly attacked and consumed the first control and one or more bugs. When attacked, the bugs emitted a strong odor. Within 7 min of the beginning ofthe trials, the anoles became more hesitant and eventually ignored S. c. cinctus but readily attacked and consumed controls. The anoles clearly discriminated between controls and S. c. cinctus; they quickly approached, attacked, and consumed controls, but ignored S. c. cinctus, even when the bugs crawled under the head or over the feet of the lizard. In a second feeding trial two to three days after the first, two of the anoles refused to attack, and two ate one S. c. cinctus each, then refused to attack subsequent bugs; yet, all anoles eagerly attacked and consumed numerous crick­ ets. 1958 KRALL ET AL. J: CH 3 -+J) CH37/\LJ)

CH 3 CH 3 (+ )-a-Pinene (-)-p-Pinene 1 3 9H c5 ~ A CH3 CH 2 CH3 CH3 p-Myrcene (+ )-Limonene Terpinolene 4 6 7

o o )lo~ ~~-H

E-2-Hexenyl Acetate E-2-0ctenal 8 9

FIG. 2. Major components from adult S. c. cinctus volatile secretions. Components 1, 3,4, 6, and 7 are found in both sexes; components 8 and 9 are found only in females.

TABLE 2. NUMBER OF ADULT BURROWING BUGS CONSUMED BY LIZARDS DURING Two TRIALS SEPARATED BY Two OR THREE DAYS

Anole Firsl trial Second lrial

I 14 I 2 6 0 3 6 1 4 1 0 BURROWING BUG SECRETIONS 1959

Charadrius vociferus and Sturnus vulgaris vs. S. c. cinctus. Both the kill­ deer and starling showed similar responses. Each readily attacked and consumed the first few (4-6) bugs offered. Then, all subsequent bugs offered (4-12) were either ignored (in spite of the bugs crawling over the bird's feet) or picked up and immediately ejected out of the beak, whereas control insects were readily eaten. Onychomys leucogaster vs. S. c. cinctus. Both mice behaved in a similar manner. The first two bugs offered were hesitantly grabbed, bitten into, released, recaptured, and then eaten. The next three bugs offered were quickly grabbed and eaten. Throughout these feeding trials the bug's secretion was readily notice­ able.

Source ofSecretion Upon pertubation of adults, secretion was observed originating from the metathoracic gland opening for both sexes. Furthermore, the characteristic odor was evident on the filter paper sliver placed on the metathoracic gland opening but not on the filter paper squares placed on the mouth or anus.

DISCUSSION

Our results suggest that the volatile secretion produced by Sehirus cincrus cinctus functions in antipredator defense: (1) The chemicals are secreted in response to disturbance. (2) Both sexes produce the major constituents, sug­ gesting that they are not used as sexual pheromones. (3) The primary constit­ uents (a-pinene, iJ-pinene, and limonene) are found in other defensive secretions and are known to have deterrent, irritating, or toxic qualities (Baker and Walmsley, 1982; Smith, 1963). (4) The bugs exhibit features of the chem­ ical defense syndrome (Whitman et at., 1985) such as aposematic coloration, gregariousness, and poor locomotory capability. For example, when attacked, they do not readily fly or run away; their primary escape defense is to drop to the ground. (5) Anoles and birds rejected them after consuming only a few. (6) Anoles developed strong food aversion conditioning after only one feeding trial. The only predators that did not show an aversion to feeding on S. c. cinctus were grasshopper mice, which each consumed the five bugs offered. The large body mass of the mice and their eagerness to consume the bugs (the speed of consumption increased with subsequent prey) led us to believe that they could. probably consume many dozens of bugs during one feeding (a limited supply of bugs prevented us from testing this). In contrast, the anoles and birds clearly exhibited increasing signs of aversion with each bug consumed. It is common for insect defenses to function differently against different predator taxa (Whit­ man, 1988; Whitman et al., 1990). In addition, Onychomys species are known 1960 KRALL ET AL. to be indiscriminate insectivores, attacking a wide mnge of toxic or otherwise defended prey including scorpions, centipedes, darkling beetles, lubber gmsshoppers, etc. (McCarty, 1978; Whitman et al., 1985). Five compounds were found in the secretion from both sexes [(IR)-( +)­ a-pinene, (lS)-(- )-,B-pinene, ,B-myrcene, (R)-( +)-limonene, and terpinolene]. a-Pinene and ,B-pinene together comprised the major portion of the secretion volatiles (91 % of GC peak area). These two compounds have been found in other hemiptemn groups such as (Hamilton et al., 1985), Lygae­ idae (Olagbemiro and Staddon, 1983), and (Aldrich et al., 1990). However, the combined concentmtion of these two compounds typically com­ prises no more than 10-20% of the total secretion (Hamilton et al., 1985; Olagbemiro and Staddon, 1983). These two compounds have also been found in termites (Baker and Walmsley, 1982). Terpenes, as a group, are fairly common in the secretions of Hemiptem (Aldrich, 1988). In addition, a reduviid bug, Apiomerus jlaviventris, uses ter­ penoids to protect its eggs (Eisner and Meinwald, 1987). The insect gathers resins from plants and coats its eggs with the resins, which contain repellent terpenoids (borneol, camphor, 1,8-cineole, etc.). The roles of two additional compounds [(E)-2-octenal, and (E)-2-hexenyl acetate] that were present only in females are unknown. Female-specific com­ pounds are found in other heteropterans (Scales, 1968; Boivin and Stewart, 1982) where they are thought to seIVe as sexual pheromones. Indeed, in Sehirus bicolor (L.), the secretion is reported to be expelled during courtship (South­ wood and Hine, 1950). Furthermore, since S. c. cinctus aggregate, one or both of these compounds may function as an aggregation pheromone. It is not known how the various secretion components of S. c. cinctus function in defense. Many chemically defended insects possess two allomonal systems that seIVe intemctive, but divergent roles: (1) distasteful, toxic, or emesis-producing compounds are held in the body; and (2) volatile compounds are secreted (Whitman et al., 1990). The chemical composition of these two systems can be quite different. Internal allomones are often large, nonvolatile compounds, whereas defensive secretions are usually mixtures ofhighly volatile, reactive, low-molecular-weight compounds (Blum, 1981). In such insects, the internal compounds produce toxicosis and elicit food aversion conditioning in predators, whereas the volatile secretion acts to startle or deter some predators, or functions in chemical aposematism (Eisner and Grant, 1981). For some predators, the defensive secretion is not deterrent by itself, but becomes deterrent when it becomes associated with the internal toxin via classical Pavlovian con­ ditioning (Brower, 1984). Some aspects of our predator tests suggest that a scenario similar to the latter might be the case in S. c. cinctus. First, no predators rejected the first S. c. cinctus they encountered, suggesting that the odor or taste of the defensive secretion was not enough, by itself, to deter consumption. It BURROWING BUG SECRETIONS 1961 was only after consumption that aversion developed, suggesting the possible presence of a slower-acting internal toxin or alternatively, an internal response to the secretion. Mint plants are notoriously rich in terpenoid compounds, and hence, S. c. cinctus may have obtained its secretion components from the seeds they feed on. However, none of the five major terpenes found in the secretions of S. c. cinctus was found in the P. vulgaris seeds. This suggests that S. c. cinctus synthesizes these chemicals de novo, modifies related precursors, or sequesters the terpenes from the mint leaves, not seeds. The life cycle of burrowing bugs is predominantly spent on or near the ground (Sites and McPherson, 1982) where these bugs may frequently encounter ants that may pose a threat to individuals or females guarding their brood. Eisner et al. (1976) have shown that a- and ,B-pinene act as irritants to ants, causing scratching and grooming reflexes that subsequently spread these compounds over the surface of the ants. We are in the process of examining the potential effec­ tiveness of the defensive secretion of S. c. cinctus against ant predators.

Acknowledgments-We wish to thank Dr. Jeffrey Aldrich; USDA-ARS. for helpful comments.

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Supplied by U.S. Dept. of Agric., National Center for Agricultural utilization Research, Peo~ia, IL