Journal of Chemical Ecology, Vol. 8, No. 3, 1982

SEX PHEROMONE COMMUNICATION IN THE ODD BEETLE, Thylodrias contractus MOTSCHULSKY (COLEOPTERA: DERMESTIDAE)

JAMES

W. M E R T I N S

Department of Entomology Iowa State University Ames, Iowa 50011 (Received June 12, 1981; revised August 10, 1981) Abstract Adult virgin females of the odd beetle, Thylodrias contractus, produce a volatile sex pheromone that influences the behavior of conspecific males and attracts them in laboratory olfactometer tests. The active substance can be collected by allowing virgin females to walk on absorbent paper disks. Disks exposed to mated females do not elicit a response from males. The male odd beetle repeatedly rubs the antennae, head, and thorax of a female with a setate glandular area on his second abdominal sternum during courtship, probably secreting a reciprocally active aphrodisiac substance. Key Words Sex pheromone, aphrodisiac, odd beetle, Thylodrias contractus Motschulsky, Coleoptera, Dermestidae, bioassay, behavior, exocrine gland.

INTRODUCTION T h e p r e s e n c e a n d a c t i v i t y o f f e m a l e - p r o d u c e d sex p h e r o m o n e s in d e r m e s t i d beetles was first d e m o n s t r a t e d f o r m e m b e r s o f the g e n e r a A t t a g e n u s a n d T r o g o d e r m a ( B u r k h o l d e r a n d D i c k e , 1966). A d u l t f e m a l e s o f s o m e A n t h r e n u s a n d D e r m e s t e s species also e m i t sex p h e r o m o n e s ( B u r k h o l d e r et al., 1974; A b d e l - K a d e r a n d B a r a k , 1979; S h a a y a , 1980), a n d t h e r e is little r e a s o n to d o u b t t h a t o t h e r m e m b e r s o f this d e s t r u c t i v e f a m i l y of beetles also use o l f a c t o r y c o m m u n i c a t i o n b e t w e e n the sexes. D u r i n g a s t u d y ( M e r t i n s , 1981) o f the life h i s t o r y , b i o l o g y , a n d b e h a v i o r o f the o d d beetle, T h y l o d r i a s c o n t r a c t u s M o t s c h u l s k y , I o b s e r v e d t h a t , w h e n 1journal Paper No. J-'10248 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011. Project No. 2259. Received for publication June 12, 1981. 653 0098-0331 / 82/0300-0653503.00/0 9 1982 Plenum Publishing Corporation

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a virgin female was placed into a container with one or more males, there was an almost immediate increase in male activity, followed by rapid orientation to and location of the female. This paper is a report on several experiments and observations of the mating behavior of T. contractus supporting the presence of sex pheromone communication in this species. METHODS AND MATERIALS

The insects used were from a laboratory culture maintained since 1973 under ambient room temperature, light, and humidity contiditions in tightfitting plastic rearing containers. The larval diet consisted of dried insect cadavers, primarily adult greater wax moths, Galleria mellonella (g.), or bits of cooked, dried beef liver. Insects for observation were removed from the cultures in the pupal stage and isolated individually in No. 3 gelatin capsules to ensure virginity. They were observed daily until adult emergence. Activities and mating behavior of individual pairs of odd beetles were observed with a dissecting microscope in an arena consisting of the bottom of a 5-cm (OD) disposable plastic Petri dish (Falcon Plastics, Oxnard, California) lined with a suitably sized disk of Whatman No. 1 filter paper. Individual virgin females were removed from gelatin capsules immediately or shortly after eclosion and held for various periods (12-120 hr) in 15-mm (OD) • 45-mm polyethylene-capped glass vials, one female per vial, with highly absorbent paper disks 2 on the bottoms. Control vials without insects and a series containing virgin males were likewise instituted and handled at the same time. For another series of tests, a group of mated females were similarly treated immediately after copulation. I investigated the presence and activity of the female sex pheromone primarily with the glass desiccator olfactometer and a bioassay similar to that described in detail by Burkholder and Dicke (1966). Briefly, the olfactometer consisted of a standard 125-mm-diameter glass desiccator, with the top replaced by a round glass plate, 6. 5 m m thick. A 20-mm diameter hole in the center of the plate allowed introduction of test insects through a small inverted glass funnel arrangement. Three other holes, each 35 m m from the center of the plate and each 15 m m in diameter, were drilled equidistant from each other. These allowed insertion of the test disks by means of glass-rodand-insect-pin holders. The test arena within the desiccator was a 10-cm-diam circle of W h a t m a n No. 1 filter paper held in a glass-walled ring. Absorbent paper disks exposed to virgin females, mated females, or males were tested against individual virgin males in the olfactometer by comparison with control disks. The exposure of an individual contaminated disk (along with its two uncontaminated controls) to an individual male constituted one test. 2No. 740-E antibacterial assay disks, 12.7-mm, Carl Schleicher and Schuell Co., Keene, New Hampshire.

SEX PHEROMONE IN DERMESTID BEETLE

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The response of the male was recorded five times at 1-min intervals after the beginning of the test. The response was designated positive if the male was within the one third of the test area containing the contaminated disk and negative if he was within the other two thirds. The test was considered positive if three or more of the five interval observations were positive and the male showed definite orientation to the contaminated disk. Tests were conducted during the months of February through June under ambient laboratory environmental conditions (21 +_ I~ 40 _+ 20% relative humidity) between 0900 and 1700 CST. Observations of the abdominal sternal gland on the male were made using standard histological techniques, including fixation in modified Carnoy's solution, staining with Delafield's hematoxylin and eosin, and microscopic examination at 1000• (Mertins and Coppel, 1971).

RESULTS AND DISCUSSION

A solitary male odd beetle in a plastic Petri dish usually is quiescent most of the time, resting with the body near and parallel to the filter paper substrate. The legs are spread out widely, and the antennae are positioned back against the thorax but elbowed forward at the 7th joint. Within 1-10 sec after addition of a virgin female to the dish, the male typically rouses f r o m lethargy, straightens his antennae, and actively moves about the arena, sometimes rapidly and directly toward the female. The anterior part of the insect is elevated at ca. 30 ~, and the antennae are spread forward and upward, slowly waving from side to side. When a female was introduced to the arena at the m a x i m u m possible distance from the resident male ( N = 34 pairs), the average elapsed time between introduction and first contact by the male was 70.4 + 67.7 (SE) sec with a range of 3-300 sec. Orientation of the male to the female is primarily by chemical stimuli evidently through an olfactory sense in the antennae. This was easily demonstrated in a preliminary experiment in which a femalecontaminated paper disk was introduced to a Petri dish arena containing 50 virgin males (Figure 1); all but three moribund males were on the disk within 20 sec. Males frequently attempted to copulate with the disk and with each other. Another preliminary experiment tested the response of males in the olfactometer to paper disks exposed to unmated females for successive 12-hr periods during the first 60 hr after eclosion (Table 1). Male response to the disks was minimal until the female was at least 36 hr old. Therefore, subsequent tests used only disks exposed to females for at least 48 hr after emergence. A total of 45 different virgin females was tested against 35 different males in 71 separate olfactometer tests. The male response was considered positive

FIG. 1. Fifty male Thylodrias contractus in a 5-cm Petri dish arena before (left) and 20 see after (right) addition of a paper disk exposed for 24 hr to a 48-hr-old virgin female.

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SEX PHEROMONE IN DERMESTID BEETLE T A B L E 1. M A L E RESPONSE IN O L F A C T O M E T E R TESTS OF P A P E R DISKS EXPOSED TO U N M A T E D F E M A L E O D D BEETLES DURING SUCCESSIVE

12-hr PERIODS Virgin female held on disk during designated hours after eclosion

No. c~c~showing + resonse No. 8c~ showing - response

0-12

12-24

24-36

36-48

48

0 6

1 7

3 10

13 4

10 2

in 55 tests and negative in 16. Response was unaffected by time of the day. By chi-square analysis, with P = 0.05 as the limit of significance, the response of males to female-contaminated disks was significantly different from the controls ( X 2 = 62.1, df = 2, P < 0.001), indicating a very strong attraction to the female pheromone. Nine of the 16 negative tests involved females that later proved to be unthrifty and short-lived, infertile, or both. In the olfactometer, males usually responded to the active disk with behavior much like that in Petri dishes previously described, concentrating their activities directly under the disk. However, because the disk was suspended out of reach 5 mm above their substrate, males inclined their bodies at a greater angle from the horizontal, extended their antennae vertically, and frequently reached toward the disk with their prothoracic legs. Concurrently, males often tried to copulate with the substrate under the disk and, when strongly stimulated, sometimes fell over backwards in the attempt. Activity of the pheromone-contaminated disks was persistent when they were held in the polyethylene-capped vials. One such disk showed no loss of activity when tested 57 days after female contamination; another still retained some activity after 16 months. An experiment similar to the preceding series used eight different mated females and eight different males in 17 separate olfactometer tests. The male response was considered weakly positive in two tests and negative in 15, suggesting that male orientation to the female-contaminated disks was not significantly different from the controls (X 2 = 3.6, df= 2, 0.05 < P < 0.20). Evidently, female pheromone activity disappears at the time of mating. This observation is supported further by the fact that males failed to respond to mated females. A series of tests involving male-contaminated disks found no positive male responses when compared with control disks. Blanchard (in Slosson, 1908) was the first to note and illustrate the "lobelike pubescent tumidity" posteromedially on the second abdominal sternum of the male odd beetle. The structure is mentioned also by Hint0n (1945) and Robert (1947) without further comment. This medial transverse swelling of the integument is more lightly colored than the surrounding sternite and bears

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a 0.3-mm-wide, dense, brushlike tuft of setae that project posteroventrally (Figures 2 and 3). My unpublished observations of the courtship and mating behavior of T. c o n t r a c t u s suggested the possibility that this structure might be involved in such activities, perhaps as a source of a chemical pheromone. During courtship the male mounts the female dorsally and orients his body in the same direction as hers. He then moves anteriorly until the setate prominence of his second abdominal sternum is above the head and uplifted antennae of the female. There he proceeds to rub the brush back and forth and occasionally sideways on the female, for ca. 1 min, sometimes moving the activity backward onto her thorax before attempting copulation. Histological examination of the structure (Figure 4) disclosed a discrete closely packed mass of presumably secretory cells beneath the setate cuticle. The cells are vacuolate, dark-staining, and contain large round nuclei. The cuticle of the second sternite is thick and sclerotized except in the area immediately over the glandular tissue, where it is thinner and without color.

FIG. 2. Adult male Thylodrias contractus (ventral view) showing the setate glandular structure on the 2nd abdominal sternum (bar = 0.5 mm).

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FIG. 3. Adult male Thylodrias contractus (ventral view) showing more closely the setate glandular structure on the 2nd abdominal sternum (bar = 0.1 mm). Numerous setae ca. 0.06 m m long occur densely in this area and fully penetrate the cuticle basally. Delicate filamentous structures seem to penetrate further into the cell mass of the gland from the bases of the setae. The results of these observations and experiments closely parallel those cited by Burkholder and Dicke (1966) and by Burkholder et al. (1974) as evidence of female sex pheromones in three other genera of dermestids. Sexpheromone-releasing or calling behavior such as that reported for Trogoderma, Attagenus, Anthrenus, and Dermestes ( H a m m a c k et al., 1976; Cross et al., 1977; Barak and Burkholder, 1977; Burkholder et al., 1974; AbdelKader and Barak, 1979) was not observed in female Thylodrias. Behavioral and anatomical evidence does exist, however, for the reciprocal production of a pheromone by the male odd beetle. It is possible that this chemical may serve as a recognition signal or aphrodisiac to the female. Alternatively, deposition

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Fro. 4. Sagittal section (8 #m) through the ventral abdominal gland of a male Thylodrias contractus (bar = 10/zm). o f such a substance could serve to m a r k a female as a l r e a d y i n s e m i n a t e d or to m a s k the f e m a l e - p r o d u c e d sex p h e r o m o n e , thus eliminating interference f r o m superfluous males d u r i n g her o v i p o s i t i o n period. Sexually d i m o r p h i c setate structures that seem to be a n a l o g o u s have been described in m e m b e r s of several beetle families ( H a l s t e a d , 1963), including D e r m e s t i d a e (Dermestes spp.)o S o m e of these have been a s s o c i a t e d with p h e r o m o n e p r o d u c t i o n (Levinson et al., 1978, 1980; Faustini, et al., 1981). Acknowledgernents--I wish to thank W.E. Burkholder, D.L. Faustini, and M.G. Klein for reading the manuscript and for many helpful suggestions. W.E. Burkholder also was kind enough to allow use of his olfactometer.

REFERENCES ABD~L-KADE~,M.M., and BA~AK,A.V. 1979. Evidence for a sex pheromone in the hide beetle, Dermestes maculatus (DeGeer) (Coleoptera: Dermestidae), J. Chem. Ecol. 5:805-813.

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BARAK, A.V., and BURKHOLDER,W.E. 1977. Behavior and pheromone studies with Attagenus elongatulus Casey (Coleoptera: Dermestidae). J. Chem. Ecol. 3:219-237. BURKHOLDER, W.E., and DICKE, R.J. 1966. Evidence of sex pheromones in females of several species of Dermestidae, J. Econ. Entomol. 59:540-543. BURKHOLDER, W.E., MA, M., KUWAHARA,Y., and MATSUMURA,F. 1974. Sex pheromone of the furniture carpet beetle, Anthrenus flavipes (Coleoptera: Dermestidae). Can. Entomol. 106:835~839. CROSS, J.H., BYLER, R.C., SILVERSTEIN, R.M., GREENBLATT, R.E., GORMAN, J.E., and BURKHOLDER,W.E. 1977. Sex pheromone components and calling behavior of the female dermestid beetle, Trogoderma variable Ballion (Coleoptera: Dermestidae). J. Chem. Ecol. 3:115-125. I~AUSTINI,D.L., BURKHOLDER,W.E., and LAUB, R.J. 1981. Sexually dimorphic setiferous sex patch in the male red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): Site of aggregation pheromone production. J. Chem. Ecol. 7:465-480. HALSTEAD, D.G.H. 1963. External sex difference in stored products Coleoptera. Bull. Entomol. Res. 54:119-134. HAMMACK, L., MA, M., and BURKHOLDER,W.E. 1976. Sex pheromone-releasing behavior in females of the dermestid beetle, Trogoderma glabrum. J. Insect Physiol. 22:555-561. HINTON, H.E. 1945. Monograph of the Beetles Associated with Stored Products, Vol. 1. British Museum, London. LEVINSON, A.R., LEVINSON, H.Z., and FRANCKE,W. 1980. Intraspecific attractants of the hide beetle, Dermestes maculatus (De Geer). XV1. Int. Congr. Entomol. ( Kyoto) Abstr.: 13R-3(9):376. LEVINSON, H.Z., LEVINSON,A.R., JEN, T.-I., WILLIAMS,J.L.D., and KAHN,G. 1978. Production site, partial composition and olfactory perception of a pheromone in the male hide beetle. Naturwissenschaften 65:543-545. MERTINS,J.W. 198 I. Life history and morphology of the odd beetle, Thylodrias contractus. Ann. Entomol. Soc. Am. 74:576-581. MERTINS, J.W., and COPPEL, H.C. 1971. Sternal gland in Coptotermesformosanus (Isoptera: Rhinotermitidae). Ann. Entomol. Soc. Am. 64:478-480. ROBERT, A. 1947. Un Dermestid~ nouveau pour ta province de Quebec. Nat. Can. (Qubbec) 74:184-194. SHAAYA,E. 1980. Sex pheromone of Dermestes maculatus DeGeer (Coleoptera, Dermestidae). J. Stored Prod. Res. 17:13-16. SLOSSON, A.T. 1908. A bit of contemporary history. Can. Entomol. 40:213-219.