Journal of Chemical Ecology, Vol. 13, No. 7, 1987

FEMALE SEX PHEROMONE OF THE COMMON FURNITURE BEETLE Anobium punctatum (COLEOPTERA: ANOBIIDAE): Extraction, Identification, and Bioassays

P E T E R R. W H I T E

and M A R T I N

C. B I R C H

Department of Zoology South Parks Road, Oxford OX1 3PS, U.K. (Received August 25, 1986; accepted October 15, 1986) Abstract--Observations and reports on the common furniture beetle Anobium punctatum suggested that, on emergence, females use a sex pheromone to attract males. GLC analysis of ovipositor extracts showed the presence of a single component, which was found to be active by EAG and coupled GLCEAG techniques, and to attract males in both walking and flying assays. The pheromone was identified by GC-MS as 2,3-dihydro-2,3,5-trimethyl-6-(lmethyl-2-oxobutyl)-4H-pyran-4 one (stegobinone), which is the sex pheromone of another anobiid, the drugstore beetle, Stegobium paniceum. Male A. punctatum responded equally to ovipositor extracts of either species, at both the sensor)' (EAG) and behavioral levels, which poses the question as to how species specificity in mate attraction is achieved. Key Words--Common furniture beetle, Anobium punctatum, drugstore beefie, Stegobium paniceum, Coleoptera, Anobiidae, sex pheromone, stegobinone, 2,3,dihydro-2,3,5-trimethyl-6-(1-methyl-2-oxobutyl)-4H-pyran-4-one, behavior, electroantennogram, species specificity.

INTRODUCTION Anobium punctatum de G e e r , the c o m m o n furniture beetle, is a w i d e s p r e a d and destructive pest o f s e a s o n e d w o o d . I n d i g e n o u s to northern E u r o p e and Asia, it has b e e n distributed by m a n on a w o r l d w i d e scale and is n o w found in Australasia, South Africa, and the eastern coast o f N o r t h A m e r i c a (Hickin, 1963). T h e d a m a g e is d o n e by the l a r v a e - - k n o w n as w o o d w o r m - - w h i c h can feed on soft and h a r d w o o d s , pulpboards, p l y w o o d , and e v e n cardboard, and as a result, both structural timbers and furniture are subject to attack. T h e f e m a l e 1695 0098-0331/87/0700 1695505 00/0 (~) 1987 Plenum Publishing Corporation

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lays her eggs in cracks in the surface, and the larvae tunnel into the wood. After pupation, the adults cut their way out, leaving a characteristic flight hole (Hickin, 1963). Surprisingly, despite the economic importance of A. punctatum, little attention has been paid to its reproductive behavior or that of anobiid beetles in general. A recent review (Tumlinson, 1985) listed only two identified pheromones from this family: the drugstore beetle, Stegobium paniceum L. (Kuwahara et al., 1975, 1978) and the cigarette beetle, Lasioderma serricorne F. (Chuman et al., 1979). Several observations, however, have suggested the presence of a female sex pheromone in A. punctatum. Kelsey et al. (1945) reported "swarming," and on examining a swarm found it to contain 167 males clustered around three females! R.W. Berry reported that males, which emerge first, locate females as they emerge from their flight holes. Our own observations have noted female "calling" behavior, which has previously been reported in A. punctatum (Cymorek, 1964) and in another anobiid beetle--Ptilinus pectinicornis L. (Cymorek, 1960): The female stands with the abdomen raised and periodically protrudes the ovipositor. This paper describes the extraction, identification, and bioassay of the sex pheromone of A. punctatum and provides comparisons with other anobiid pheromones. METHODS AND MATERIALS

Insect Material. Cultures of hazel twigs infested with A. punctatum larvae and pupae were obtained from the Building Research Establishment, Princes Risborough, Buckinghamshire, U.K., and were kept at 22~ in constant darkness. Emerging adults were removed daily and sexed using the presence or absence of a depression on the ventral surface of the male's terminal abdominal segment (Kelsey et al., 1945). The sexes were kept separately in glass tubes on moist filter paper until use. S. paniceum was kept in a mixed-sex culture on rabbit pellets at 22 ~ Extraction Procedure. Ten newly emerged female A. punctatum were dissected and the ovipositors removed and extracted in 0.5 ml of pentane for 24 hr at 4~ Three replicate extractions were made. Ovipositors of S. paniceum were extracted in a similar manner. Gas Chromatography. Both extracts were analyzed using a Varian 3700 GLC (Walton-on-Thames, Surrey, U.K.) fitted with a 50-m x 0.32-ram ID fused silica column coated with chemically bonded OV-1 (Phase Separation Ltd. Queensferry, Clwyd, U.K.). Samples were injected in splitless mode with the oven at 50 ~ C for 1 min, then programmed at 5~ to 250~ using helium carrier gas at 1.5 kg/cm 2. Quantitative analyses used naphthalene as a standard.

COMMON FURNITURE BEETLE PHEROMONE

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Gas Chromatography-Mass Spectrometry (GC-MS). GLC of the A. punctatum and S. paniceum extracts each showed the presence of a single major component which was identified using a Finnigan 1020 GC-MS. Electroantennography (EAG). Initial EAG recordings showed that male A. punctatum antennae gave a strong EAG response to the conspecific ovipositor extract. An EAG experiment was therefore conducted to determine whether males and females are equally responsive to female A. punctatum extract, and whether they respond differently, at the EAG level, to extracts of A. punctatum and S. paniceum. EAG responses were recorded from isolated furniture beetle heads mounted on a cork stage using double-sided adhesive tape. The indifferent glass microelectrode was inserted into the base of the head, and the opening sealed with petroleum jelly. The recording microelectrode was inserted into a small hole punched in the terminal antennal segment using a finely etched tungsten needle. Electrodes were filled with saline and connected, using chloridized silver wires, to a Grass P-16 DC (Quincy, Massachusetts) preamplifier and a Tektronix 5223 oscilloscope. The odor delivery system was similar to that described by Payne and Dickens (1976), using a two-way solenoid valve to divert the filtered (activated charcoal) and humidified airflow via a test cartridge into a glass Y delivery tube. The airflow was maintained at 1000 ml/min with a pulse length of 1 sec. Cartridges consisted of a strip of filter paper impregnated with 10/zl of the test extract, inside a Pasteur pipet. Parallel series of dilutions of female A. punctatum and S. paniceum extracts were made, giving matched pairs containing equal concentrations of the major component. The two series were checked for concentration by GLC and stored at - 8 0 ~ before use. Test cartridges were presented pairwise in ascending order of concentration. Control presentations of solvent were interspersed between test presentations, and the response subtracted from the test response before analysis of the results. Six individuals of each sex of A. punctatum were tested in this way. Results were analyzed using a split-plot analysis of variance to identify differences between the sexes and between the responses to the two extracts. Coupled Gas Chromatography-Electroantennography (GC-EAG). This was carried out to confirm that the EAG responses recorded to the ovipositor extracts were due to the single major component rather than to any trace components. The GC-EAG system was essentially similar to that described for GCsingle cell recordings by Wadhams (1984). The GLC and OV-1 column were as described above, using a temperature program of 150-230~ at 5~ The column effluent was split 1 : 1 between two lengths of an OV-l-coated capillary column using a glass lined T piece. One arm led to the FID detector while the other ran via a heated jacket (maintained at 220~ to the airflow impinging on the antenna. Behavioral Assays. Bioassays were conducted to determine the response of male and female A. punctatum to conspecific ovipositor extract and to test

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~

filter paper arena

glass tube

lOcmring

ffl

dia (Ruon coated)

cover

i

I

~

test disc

F tuon coating

contro[ disc

Fio. 1. Two-choice pitfall bioassay apparatus.

whether the response of A. punctatum to this extract differed from that to S. paniceum ovipositor extract. A two-choice pitfall assay was used to compare the response of the sexes (Figure 1). Two 5 x 2.5-cm glass tubes with necks coated in Fluon were glued below holes cut in an 11-cm-diameter filter paper disc, which was supported on a metal baseplate. A 10-cm-diameter Perspex ring coated with Fluon was placed on the filter paper and covered with a glass plate. The insects (3-15 per test) were introduced into the arena and allowed 10 min to settle. Test and control stimuli were then introduced, consisting of 10 /zl of female extract (0.2 female equivalents), and 10 /A of pentane, respectively, applied to a 1-cm filter-paper square. The assay was left for 1 hr at 25~ and then the number of beetles in each tube was counted. Five replicates were conducted for each sex, using each individual once only. Results were combined to give totals of each sex trapped in the test or control tubes. Differences between test and controls were tested for significance using a chi-square test. A preliminary assay was also conducted in a wind tunnel 160 cm long x 75 cm high x 70 cm wide, using a wind speed of ca. 0.2 m/sec. The test stimulus (0.4 female equivalents of ovipositor extract) was placed on a filter paper strip at the upwind end of the tunnel, and either males or females placed on a platform 1.5 m downwind of the source, and their behavior observed. An open arena assay was employed to compare the behavioral response of male A punctatum to the two extracts. The arena again consisted of a 10-cmdiameter ring coated in Fluon placed on an 11-cm-diameter filter paper and covered by a glass plate. Males were released singly into the arena and allowed 10 min to settle. The test extract was then applied to a 1-cm filter paper square as described above, the paper placed in the center of the arena, and the arena covered. Each individual was observed for up to 5 min, and the time taken for it to reach the center o f the arena (defined as crossing a 2-cm-diameter circle drawn around the test paper) recorded. Beetles not responding in this period were given the default time of 5 rain. Three concentrations plus a control were

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tested for each of the extracts, with l0 replicates of each, in a randomized design. Results were analyzed using analysis of variance to determine whether the response time differed between the two extracts. RESULTS GLC. Analysis of the A. punctatum ovipositor extract showed the existence of a single major peak with a retention time of 27.5 min using the OV-1 column and the conditions described (Figure 2). Quantitative GLC showed that the mean (N = 10) amount of this material per female A. punctatum was 240 rig. Analysis of the S. paniceum extract also showed a single major peak with an identical retention time, which was confirmed by coinjection. GC-MS. The electron impact spectra for the major components of A. punctatum and S. paniceum ovipositor extracts were found to be identical (Figure 3). They suggested a compound of molecular weight 224, this being the highest ion of significance in both spectra. The spectra were also identical to that published for stegobinone, the sex pheromone of S. paniceum (Kuwahara et al., 1975, 1979). Therefore, on the basis of identical spectra and retention times, the major component of the A. punctatum ovipositor extract was identified as stegobinone, or 2,3-dihydro-2,3,5-trimethyl-6-(1-methyl-2-oxobutyl)-4Hpyran-4-one (Figure 4). EAG. Results of the EAG experiment (Figure 5) showed that the EAG response of males was significantly greater than that of females to both A. punctatum and S. paniceum extracts (P < 0.001). However, within each sex, there

0

3

6

9

12

15

18

21

24

27

30

45rain

FIo. 2. Gas chromatograrn of A. punctatum ovipositor extract, using 50-rn OV-1 column. Oven initially at 50~ for 1 min, then programmed at 5~ to 250~

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MASS SPECTRA 57

Anobium punctatum

168

113 B3

12~, 139

22t+

,,I, I ,.IJ

M/E

50

I00 57

250

150

Steg0bium paniceum

!68

113

Ml[

50

100

150

200

2

FIG. 3. Electron impact mass spectra of major components of A. punctatum and S.

paniceum ovipositor extracts.

COMMON FURNITURE BEETLE PHEROMONE

170 l I

0 FIG. 4. Structure of 2,3-dihydro-2,3,5-trimethyl-6-(1-methyl-2-oxobutyl)-4H-pyran-4one (stegobinone).

1,6

1,4

/'

1,2

d'

1,0 EAG 0,8 rnV 0,6

0,4

0,2

0,001

0,01

0,1

1,0

DOSE (~A.punctatum equiv.) FI~. 5. Electroantennogram (EAG) response of A. punctatum to ovipositor extracts of A. punctatum (open circles) and S. paniceum (closed circles). ( • standard error, N = 6). Using analysis of variance, difference between male and female response was F = 13.5, P < 0.001; difference between extracts F = 0.0, NS.

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GLC I

Inj

0.4 rnV

EAG

5 10 15rain FIG. 6. Coupled GC-EAG response of male A. punctatum to conspecific ovipositor extract. GC oven intially at 150~ for 1 min, then programmed at 5~ to 230~

was no significant difference between the responses to the two extracts. Coupled G C - E A G traces for male A. punctatum (Figure 6) showed a single EAG response that was synchronous with elution of the single major component of the conspecific ovipositor extract. The E A G response to the female extract was therefore due to a response to the major component and not to any trace components. Behavioral Assays. Results of the pitfall assays are presented in Table 1. Significantly more male A. punctatum were trapped by the conspecific female extract than the control at the concentration tested (P < 0.001), but there was no evidence of such a bias in females. TABLE 1. ATTRACTION OF A. punctatum TO CONSPECIFIC OVIPOSITOR EXTRACT IN Two CHOICE PITFALL ASSAY. a

Male Females

Test (0.2 FE)

Control (pentane)

No response

Total

30 0

0 2

13 29

43 31

~Difference between test and control: males X~l) = 30.0, P < 0.001; females, NS.

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A similar result was found in the wind-tunnel trials (data to be presented elsewhere). At least 20 of the 40 males tested responded by walking to the upwind edge of the platform, taking off and flying upwind, and landing on or within 2 cm of the source. Males flew to both A. punctatum and S~ paniceum extracts. By comparison, no females flew in the wind-tunnel trials. The response of males in the open arena assays (Figure 7) showed a significant effect of concentration (P < 0.001) with the time taken to reach the source decreasing with increasing dosage. Males responded to the odor by raising their antennae and then orienting in a series of zigzags to the source. Once at the source, males performed tight turning movements on the filter paper square. In contrast, when exposed to the solvent controls, males either remained stationary at the edge o f the arena, or walked slowly around the perimeter. There was no difference, however, between the response of males to the two extracts. Female responses to the ovipositor extracts were not significantly different from responses to the solvent, even at the highest concentration tested (Figure 7).

300 -

o§

[] \

\

\

\ \

200

RESPONSE TIME (sees) tO0

# Pen'tane

0,05

0,5

0,0002

0,002

5

ng DOSE

0,02

?equiv

FIG. 7. Response of A. punctatum males to ovipositor extracts of A. punctatum (open circles) and S. paniceum (closed circles) in open arena behavioral assay. Response time gives mean time (_+ standard error, N = 10) taken to reach center of arena. Using analysis of variance, effect of dose F = 40.6, P < 0.001; difference between extracts F = 0.1, NS.

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The female common furniture beetle, A. punctatum, was shown to release from the ovipositor a sex pheromone which attracts males. The GC-EAG results suggest that only one component from the ovipositor produces the response, and on the basis of mass spectra and retention times this was identified as stegobinone [2,3-dihydro-2,3,5-trimethyl-6-(1-methyl-2-oxobutyl)-4H-pyran4-one]. This molecule possesses three chiral centers, giving eight possible stereoisomers. So far we have not been able to determine directly the configuration of the stegobinone extracted from A. punctatum, although the configuration of the sex pheromone of S. paniceum has been established by X-ray analysis as (2S,3R,7R)-stegobinone (Hoffmann et al., 1981). Since male furniture beetles responded identically to the extracts from A. punctatum and S. paniceum in both behavioral and electrophysiological assays, the sex pheromone ofA. punctatum appears to have the same configuration. However, further chemical analysis is required to confirm this. Males have a low threshold for this material, responding in the behavioral assay at doses below 0.05 ng (2 • 10 - 4 female equivalents) and in the EAG assay at 0.25 ng (10 -3 FE), and attraction of males was further demonstrated in a flight tunnel. To our knowledge, this is the first instance where attraction of flying beetles to a pheromone source has been demonstrated in the laboratory. Choudhury and Kennedy (1980) reported this response in flying Scolytus multistriatus, but in uniform concentrations of pheromone. Female common furniture beetles produce large amounts of the sex pheromone. The mean titer per female was 240 ng, and since female size varied widely (probably reflecting differences in the duration of the larval stage), some females are likely to contain considerably more than this. Kuwahara et al. (1975) found similar levels of pheromone in S. paniceum (50-200 ng per female). Furthermore, pheromone titer was found to rise following emergence in both S. paniceum (Kuwahara et al., 1975) and Lasioderma serricorne (Coffelt and Burkholder, 1972), reaching a plateau after five days. In this study female furniture beetles were extracted at 1 to 3 days following emergence, so they may not have reached a peak of pheromone content. This high pheromone content may result from strong selection acting on females to ensure that they are located by males, since the adult life-span is only about two weeks after a larval development that may last four years. The results suggest that both A. punctatum and S. paniceum use the same sex pheromone. Although both species are placed in the subfamily Anobiinae, they are not generally positioned immediately adjacent to each other (White, 1971; Toskina, 1974). Crowson suggests (personal communication) that the two genera separated 40-50 million years ago in the Palaeocene, yet they appear to have retained the same sex pheromone. We intend to study other anobiid beetles to see if similar compounds are found.

COMMON FURNITUREBEETLE PHEROMONE

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Since male common furniture beetles respond equally well to the two extracts, it appears that they will be equally attracted to calling female drugstore beetles. We have yet to investigate the response of male drugstore beetles to determine whether they are able to discriminate between the two extracts, but at least in the case of A. punctatum there is the possibility of confusion if the species coexist. While other barriers to interspecific mating are likely to exist, selection should act to promote discrimination between species at the earliest possible stage, i.e., during location of the female, to avoid wasting time and energy following false trails (Linnet al., 1986; Card6 and Baker, 1984). The two species appear to have been allopatric prior to association with human habitation; A. punctatum from the forests of northern Europe and Asia (Hickin, 1963), and S. paniceum from the Mediterranean or Middle East (Crowson, personal communication). They were further separated ecologically, since the former develops in wood, while the latter is found in bird nests (Woodroffe, 1953) and stores of organic material. However, S. paniceum has become a cosmopolitan pest of warehouses, stores, and houses (Mallis, 1960) since it can develop on almost any dried organic material (Pant and Fraenkel, 1954) and is now sympatric with A. punctatum over much of its range. Therefore, at least in houses, stores, and animal buildings, the two species may coexist. It will be interesting to determine whether confusion by males between the calling females does occur under such conditions, and if so, whether discriminating mechanisms develop. Acknowledgments--We should like to thank: Mr. J. Grandidge for some assistance with bioassays; Mr. R.W. Berry, Building Research Establishment, Princes Risborough Laboratory, Bucks, U.K., for supplying insects and advice; Dr. R.T. Aplin, Oxford, and K. Rollins of V.G. Masslab, Altrincham, Cheshire, for assistance with GC-MS; Prof. F.R. Whatley, Department of Plant Sciences, Oxford, for use of GC-MS; Dr. R.A. Crowson, Department of Zoology, Glasgow, for helpful advice; and Prof. J.S. Kennedy for critical review of the manuscript.

REFERENCES

CARDt~,R.T., and BAKER, T.C. 1984. Sexual communication with pheromones, pp. 355-386 in Chemical Ecology of Insects. W.J. Bell and R.T. Card6 (eds.). Chapman and Hall, London.

COFFELT,J.A., and BURKHOLDER,W.E. 1972. Reproductive biology of the cigarette beetle, Lasioderma serricorne. 1. Quantitative laboratory bioassay of the female sex pheromone from females of different ages. Ann. Entomol. Soc. Am. 65:447-450. CHOUDHURY,J.H., and KENNEDY,J.S. 1980. Light versus pheromone-bearing wind in the control of flight direction by bark beetles, Scolytus multistriatus. Physiol. Entomol. 5:207-214. CHUMAN, T., KOHNO, M., KATO, K., and NOGUCHI, M. 1979. 4,6-dimethyl-7-hydroxynonan-3one, a sex pheromone of the cigarette beetle (Lasioderma serricorne F.). Tetrahedron Lett. 25:2361-2364. CYMOREK,S. 1960. Ober das Paamngsverhalten und zur Biologie des Holzsch~idlings Ptilinus pectinicornis L. (Coleoptera: Anobiidae). XI International Kongress Ftir Entomologie, Wien. Verhandlungen 2:335-339.

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CYMOREK,S. 1964. Beitrfige zur Kenntnis der Lebensweise und des Schandauftretens holzzerst6render Insekten. Z. Angew. Entomol. 55(1):84-93. HICKIN, N.E. 1963. The Insect Factor in Wood Decay. Hutchinson, London.

HOFFMANN,R.W., LADNER,W., STEINBACH,K., MASSA,W., SCHMIDT,R., and SNATZKE,G., 1981. Absolute Konfiguration yon Stegobinon. Chem. Ber. 114:2786-2801. KELSE'f, J.M. SPILLER, D., and DENNE, R.W., 1945. Biology of Anobium punctatum. Progress report. N.Z. Sci. Tech. Bull. 27:59-68. KUWAHARA,Y., FUKAMI,H., ISHII, S., MATSUMURA,F., and BURKHOLDER,W.E. 1975. Studies on the isolation and bioassay of the sex pheromone of the drug-store beetle, Stegobium paniceum (Coleoptera: Anobiidae). J. Chem. Ecol., 1(4):413-422. KUWAHARA,Y., FUKAMI,H., HOWARD, R., ISHII, S., MATSUMURA,F., and BURKHOLDER,W.E., 1978. Chemical studies on the Anobiidae: Sex pheromone of the drag-store beetle, Stegobium paniceum (L.) (Coleoptera). Tetrahedron 34:1769-1774. LINN, C.E., JR., CAMPBELL,M.G., and ROELOFS,W.L. 1986. Male moth sensitivity to multicomportent pheromones: Critical rote of female-released blend in determining the functional role of components and active space of the pheromone. J. Chem. Ecol. 12(3):659-668. MALLIS, A. 1960. Handbook of Pest Control. MacNair-Dorland Company, New York. PANT, N.C., and FRAENKEL,G. 1954. Studies on the symbiotic yeasts of two species, Lasioderma serricorne F. and Stegobium paniceum L. Biol. Bull. 107:420-432. PAYNE, T.L., and DICKENS, J.C. 1976. Adaptation to determine receptor system specificity in insect olfactory communication. J. Insect Physiol. 22:1569-1572. TOSKINA, I.A. 1974. A construction of the system of Anobiinae (Anobiidae: Coleoptera) by mathematical methods. Zh. Obshch. Biol. 35:297-302. TUML1NSON,J.H. 1985. Beetles: Pheromonal chemists par excellence, pp. 367-380, in P. A. Hedin (ed.). Bioregulators for Pest Control. ACS Symposium Series No. 276, Washington. WADrlAMS,L.J. 1984. The coupled gas chromatography-single cell recording technique, pp. 179189 in H.E. Hummel and T.A. Miller, (eds.). Techniques in Pheromone Research. SpringerVerlag, New York. WHITZ, R.E. 1971. Key to North American genera of Anobiidae with phylogenetic and synonymic notes (Coleoptera). Ann. Entomol. Soc. Am. 64:179-191. WOODROFFE,G.E. 1953. An ecological study of the insects and mites in the nests of certain birds in Britain. Bull. Entomol. Res. 44:739-772.