Coleoptera: Cerambycidae) of the Primitive Subfamily Prioninae

BEHAVIOR Cuticular Hydrocarbons Mediate Mate Recognition in a Species of Longhorned Beetle (Coleoptera: Cerambycidae) of the Primitive Subfamily Prioninae

1 2 2 JAMES D. BARBOUR, EMERSON S. LACEY, AND LAWRENCE M. HANKS

Ann. Entomol. Soc. Am. 100(2): 333Ð338 (2007) ABSTRACT We tested the hypothesis that contact pheromones mediate mate recognition in Prionus californicus Motschulsky, a species of longhorned beetle (Coleoptera: Cerambycidae) in the primitive subfamily Prioninae. Males attempted to mate with live females only after contacting them with their antennae, and 80% of males showed an identical response to freshly killed females. Males did not attempt to mate with dead females that had been extracted with solvent, suggesting that mate recognition cues had been eliminated. When the solvent extract was applied to carcasses of the same dead females, however, 56% of the males again attempted to mate with them. A preliminary analysis of crude solvent extracts of adult beetles revealed that adults have at least 24 different cuticular hydrocarbons and that the sexes differ in relative proportions of some compounds that may serve as the contact pheromone. This report provides the Þrst evidence that contact pheromones play an important role in mate recognition in the more primitive longhorned beetles.

KEY WORDS Prionus californicus, mating behavior, contact pheromone, chemoreception

Volatile sex pheromones have been identiÞed for sev- males recognize females by antennal contact chemo- eral beetle species of the Cerambycidae, all of which reception, and the probability of Þnding a mate is are members of the diverse subfamily Cerambycinae correlated with area search rate (Hanks et al. 1996, (for review, see Ray et al. 2006). The pheromones are Hanks 1999). All of the research on contact phero- produced by males and consist of molecules six, eight, mones of cerambycid species to date has been re- or 10 carbons in length, with hydroxyl or carbonyl stricted to species of the phylogenetically advanced groups at C2 and C3 (for review, see Lacey et al. 2004). subfamilies Cerambycinae and Lamiinae (Kim et al. Volatile pheromones produced by females have been 1993; Fukaya et al. 1996, 1997, 2000; Wang 1998, Ginzel identiÞed for two species, Migdolus fryanus Westwood and Hanks 2003, Ginzel et al. 2003a,b, 2006). To our and Verperus xatarti Dufour (Leal et al. 1994, Boyer et knowledge there has been no research on contact al. 1997), that formerly were placed in the Ceramby- pheromones of species in the more primitive subfam- cidae but whose family-level taxonomy is uncertain ilies that lack elongate antennae, including the Paran- (Napp 1994, Dong and Yang 2003). Recent studies drinae, Prioninae, and Aseminae (sensu Monne´ and have provided the Þrst conclusive evidence of a fe- Hovore 2005). Information on the use of contact pher- male-produced volatile sex pheromone for a species of omones in these primitive subfamilies would provide the primitive cerambycid subfamily Prioninae, Prionus valuable insight into the evolution of mating systems californicus Motschulsky (Cervantes et al. 2006). in the Cerambycidae. Volatile pheromones of cerambycids serve to attract In this article, we test the hypothesis that contact the opposite sex from a distance and to bring the sexes pheromones mediate mate recognition in Prionus cali- into proximity (Lacey et al. 2004). There is a growing fornicus Motschulsky (Cerambycidae: Prioninae). This body of evidence that the next behavioral step, mate species is distributed from northern Mexico to British recognition, is mediated by contact pheromones that Columbia, and it is among the largest of North Amer- are made up of long-chain cuticular hydrocarbons in ican cerambycids (adult body length 25Ð60 cm; Lin- the epicuticular wax layer of females (for review, see sley 1962). The larvae feed on the roots of at least 21 Ginzel et al. 2006). Moreover, the elongate antennae genera of woody perennials in 12 families, and they of cerambycid species, the “longhorned beetles” for can be economically important pests of woody plants which the family is named, may be adaptive because in agricultural and urban landscapes (Johnson and Lyon 1991). Females lay eggs on or in the soil near host plants, and the larvae complete development in 3Ð5 yr 1 Corresponding author: Parma Research and Extension Center, Uni- (for natural history, see Linsley 1962, Bishop et al. versity of Idaho, Parma, ID 83660-9637 (e-mail: [email protected]). 2 Department of Entomology, University of Illinois at Urbana- 1984). Adults emerge in late June and July in south- Champaign, Urbana, IL 61801. western Idaho and are crepuscular and sexually di-

0013-8746/07/0333Ð0338$04.00/0 ᭧ 2007 Entomological Society of America 334 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2 morphic: males are smaller than females and have tempt to mate was evidence that recognition cues larger and more strongly serrate antennae. Males are were intact and that a behavioral signal by the active ßiers, whereas females are sedentary and rarely female was not necessary for mate recognition. ßy. Adults apparently do not feed and have atrophied 3. Extracted female. Cuticular hydrocarbons were ex- digestive tracts; consequently, they are short lived tracted from freeze-killed females by placing them (Ϸ2 wk under laboratory conditions; J.D.B., unpub- singly into clean glass jars (wide mouth, 60 ml, lished data). SA-99535-40, Cole-Parmer Instrument Co., Vernon Hills, IL) containing 10 ml of HPLC-grade hexane (Chromasolv 270504, Aldrich, Milwaukee, WI) and Materials and Methods gently agitating for 10 min. Jars were sealed with Source of Beetles. We collected larvae of P. califor- Teßon-lined lids, and extracts were stored at Ϫ20ЊC. nicus in fall 2004 from infested hop yards in Canyon Each carcass was air-dried for 30 min to allow sol- Co., ID. Large larvae (5Ð8 cm in length) were dis- vent to evaporate before the bioassay. Lack of a lodged from uprooted hop crowns and transported to response by the male was evidence that chemical a Þeld plot at the University of Idaho Parma Research recognition cues had been eliminated. and Extension Center (Canyon Co.). We had pre- 4. Reconstituted female. Entire solvent extract (one pared 19-liter buckets by drilling two holes (0.75 cm female equivalent) pipetted slowly and evenly over in diameter) into the bottoms for drainage and Þlling the elytra of the same extracted female, solvent them with soil from the Þeld plot to within 10 cm of allowed to evaporate for 30 min. Mating response by the top. We buried one larva in each bucket (Ϸ10 cm male was evidence that chemical recognition cues in depth) with a 15Ð35-cm-long section of hop rhi- had been restored and had been isolated in the zome that provided them food. Buckets then were solvent extract. placed in a trench and buried to within Ϸ10 cm of the top of the buckets. Larvae overwintered in the Bioassays were conducted by placing a live female buckets. or treated female carcass in the arena and then intro- Hop plants growing in the buckets were pruned ducing a male. Living females were allowed to accli- back in early spring 2005 and buckets were Þtted with mate for 2 min before introducing the male, and the lids (center removed and replaced with aluminum male was removed before he could copulate with the screening) to capture emerging beetles. Water was female. After a male contacted a living female with his supplied to each bucket by drip irrigation (8 liters/h antennae, a clear progression of behaviors led to at- at 0.14 MPa) for 10 min/d. Buckets were monitored tempted copulation (see Results and Discussion): 1) daily from mid-June through the end of July, and male stops walking, 2) aligns his body with the female, newly emerged beetles were collected singly into ven- 3) mounts her, and 4) bends his abdomen to connect tilated glass jars. Males and females were held in sep- genitalia. These behavioral steps are cumulative (i.e., arate growth chambers (22ЊC and a photoperiod of a male must perform steps 1Ð3 to reach step 4). We 16:8 [L:D] h) and were unmated. Beetles used in scored the response of males to females according to bioassays were 1Ð14 d old and were active and appar- these behavioral steps. A male was scored as “no re- ently healthy. Voucher specimens have been submit- sponse” if he showed none of these behaviors after ted to the W. F. Barr Entomology Museum at the contacting the female with his antennae three times. University of Idaho, Moscow. We presented three different males to each of six Mating Behavior and Bioassays. We characterized females with the same male tested twice against each the mating behavior of P. californicus and identiÞed of the sequential female treatments (n ϭ 18 males ϫ cues involved in mate recognition by observing paired four treatments ϫ two trials ϭ 144 bioassays). females and males in a glass arena (19 cm in diameter We initially tested the response of males to treat- by 6.4 cm in height) lined with Þlter paper (Whatman ments by categorical analysis of variance (ANOVA) no. 1, Whatman, Maidstone, England). All bioassays (PROC CATMOD, SAS Institute 1999), including a were conducted in July 2006 during the second to sixth trial effect (two trials per male per treatment) and the hour of scotophase when beetles typically were active trial-by-treatment interaction. Neither the trial nor (Cervantes et al. 2006). Bioassays were conducted interaction terms were signiÞcant (see Results and under red light (25-W incandescent) to minimize the Discussion). We therefore pooled data from both tri- inßuence of visual cues, and beetles were videotaped als of each male and tested statistical signiÞcance of (Sony DCR-TRV80) to facilitate characterization of the response to treatments with Fisher exact two- mating behavior. Bioassays to evaluate the role of tailed test (Sokal and Rohlf 1995). We compared num- contact pheromones in mate recognition tested the bers of males displaying either a partial response (i.e., responses of individual males to females that had been reaching behavioral step 1 at least) or a complete subjected to the following sequential treatments: response (i.e., reaching behavioral step 4) to 1) live females versus freeze-killed females, 2) freeze-killed 1. Living female. An attempt to mate was evidence that females versus extracted females, and 3) extracted fe- behavioral and/or chemical recognition cues were males versus reconstituted females. intact. Analyses of Cuticular Hydrocarbons. A preliminary 2. Freeze killed (Ϫ4ЊC for 30 min) female, carcass analysis of cuticular hydrocarbons was conducted us- warmed to room temperature (Ϸ15 min). An at- ing whole-body extracts of Þve adult P. californicus of March 2007 BARBOUR ET AL.: MATE RECOGNITION IN PRIMITIVE LONGHORNED BEETLE 335

Lamiinae, males of which immediately attempt to mate with females after Þrst contacting them with their antennae, and females rarely if ever being re- fractory (Hanks et al. 1996; Ginzel and Hanks 2003; Ginzel et al. 2003b, 2006, and references therein). These Þndings suggest that the role of contact pheromones in mate recognition has been conserved during the evolution of the Cerambycidae. The response of male P. californicus to females was strongly inßuenced by the treatments to which females were subjected (PROC CATMOD; df ϭ 3, ␹2 ϭ 27.0, P Յ 0.0001), but neither the trial effect nor the interaction were signiÞcant (␹2 ϭ 0.06, df ϭ 1, P Յ 0.80 and ␹2 ϭ 0.53, df ϭ 3, P Յ 0.91, respectively). Males in all trials responded to living females by reaching at least behavioral step 3 (mounting), and in 35 of 36 trials (97%) reached step 4 (attempting to connect Fig. 1. Response of male P. californicus to females that were alive, freeze killed, extracted with solvent (cuticular genitalia; Fig. 1). Males in most trials mounted the hydrocarbons removed), and reconstituted (solvent extract same females after the females had been freeze killed, applied to cuticle). Behavior of responding males was cate- and the step 3 response was similar to their response gorized according to the progression of mating behaviors to living females (Fisher exact test; P ϭ 0.12). The step described in Materials and Methods. n ϭ 36 males for each 4 response to dead females (28 of the 36 trials), how- treatment. ever, was signiÞcantly lower than that for living females (P ϭ 0.027) (Fig. 1), indicating that behavioral signals from females, or their body position, may in- each sex. Beetles were individually freeze killed, and ßuence the mating response by males. cuticular hydrocarbons were extracted as described When presented with extracted females, only seven above. Extracts were analyzed by coupled gas chro- males reached step 1, and none progressed further matography (GC)-mass spectrometry with electron (signiÞcantly different from the step 1 response to impact ionization (70 eV) by using a HewlettÐPackard freeze-killed females; P Ͻ 0.0001). This Þnding con- (HP) 6890 GC (HewlettÐPackard, Sunnyvale, CA) Þrms that mate recognition cues had been removed by equipped with a DB-5MS capillary column (30 m by 0.25 mm by 0.25 ␮m Þlm; J&W ScientiÞc, Folsom, CA) -in splitless mode interfaced to an HP5973 mass selec- Table 1. Relative concentrations (% of total ؎ 1 SD) of hy drocarbon compounds in crude extracts of adult female and male (per sex 5 ؍ tive detector, with helium carrier gas. The GC was P. californicus (n programmed from 50ЊC/1 min, 10ЊC/min to 240ЊC, and then to 300ЊCat2ЊC/min, held 5 min. Injector and Retention Relative abundance Peak transfer line temperatures were 280ЊC. Differences time (mean Ϯ SD) P no. between the sexes in mean relative proportions of (min) Females Males speciÞc hydrocarbons were tested using a two-tailed 1 23.56 0.05 Ϯ 0.07 0.55 Ϯ 0.30 0.006 t-test. 2 23.80 Not detected 0.07 Ϯ 0.08 3 24.10 Not detected 0.12 Ϯ 0.13 4 24.34 0.61 Ϯ 0.36 1.14 Ϯ 0.51 ns Results and Discussion 5 24.49 Not detected 0.23 Ϯ 0.10 6 24.70 Not detected 0.13 Ϯ 0.14 All of the male P. californicus attempted to mate 7 25.09 33.0 Ϯ 1.6 28.4 Ϯ 3.00 0.018 with females in laboratory trials, and all but one male 8 25.36 4.68 Ϯ 1.70 6.55 Ϯ 1.20 ns repeated this response in the second trial. All of the 9 25.59 1.39 Ϯ 0.42 2.40 Ϯ 0.48 0.007 10 25.83 22.6 Ϯ 1.90 20.2 Ϯ 3.60 ns live females were receptive to mating. In every case in 11 26.01 0.42 Ϯ 0.39 1.03 Ϯ 0.24 0.018 which a male attempted to mate with a living female, 12 26.06 3.13 Ϯ 0.42 2.88 Ϯ 0.66 ns he responded only after contacting her with his an- 13 26.13 0.65 Ϯ 0.78 1.77 Ϯ 0.39 0.021 tennae. Males did not seem to approach females di- 14 26.20 0.19 Ϯ 0.17 0.26 Ϯ 0.31 ns 15 26.50 24.1 Ϯ 1.40 13.6 Ϯ 1.80 0.001 rectly in a manner that suggested they were respond- 16 26.70 Not detected 0.24 Ϯ 0.27 ing to visual or auditory cues, or to volatile attractants. 17 26.75 2.96 Ϯ 0.94 4.92 Ϯ 1.00 0.014 This Þnding suggests that males did not use the volatile 18 26.93 0.56 Ϯ 0.77 6.89 Ϯ 0.78 0.001 pheromone (Cervantes et al. 2006) to locate females 19 27.04 0.83 Ϯ 0.29 Not detected 20 27.18 2.26 Ϯ 0.68 4.44 Ϯ 0.55 0.001 in the still-air conditions of the bioassay arena. Once 21 27.35 1.95 Ϯ 1.40 2.82 Ϯ 0.95 ns a male touched a female with his antennae, he stopped 22 27.89 0.43 Ϯ 0.34 0.32 Ϯ 0.32 ns walking, aligned his body to hers, and immediately 23 28.40 Not detected 0.10 Ϯ 0.10 mounted her, grasping her pronotum or elytra with his 24 28.70 0.25 Ϯ 0.32 0.95 Ϯ 0.65 ns forelegs. The male then bent his abdomen to connect Peak numbers correspond to peaks in Fig. 2. Statistical signiÞcance with the femaleÕs claspers. These behaviors are very of differences between the sexes in mean relative abundance was similar to those of species in the Cerambycinae and tested with two-tailed t-tests (ns, not signiÞcant; P Ͼ 0.05). 336 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 2

Fig. 2. Representative total ion chromatograms of whole-body hexane extracts of adult female (top) and male (bottom) P. californicus. Analysis conditions are described in Materials and Methods. March 2007 BARBOUR ET AL.: MATE RECOGNITION IN PRIMITIVE LONGHORNED BEETLE 337 washing the females in solvent. Males responded strongly to reconstituted females, however, with 35 of 36 males reaching step 1 (similar to their response to freeze-killed females; P ϭ 0.99) and 20 males (56%) reaching step 4 (also not signiÞcantly different from response to freeze-killed females; P ϭ 0.079). This response to reconstituted females con- Þrms that mate recognition cues had been largely restored by applying extract to females and that the cue was isolated in the extract. The reduction in the response of males to reconstituted females compared with freeze-killed females, although not statistically signiÞcant, suggests that some activity was lost during extraction and reapplication of cuticular hydrocarbons. Males may not have responded as strongly to reconstituted females because the process of solvent extraction and reapplication of hydrocarbons changes Fig. 3. Male P. californicus attempting to mate with a glass the natural structure of the wax layer, as was appar- stopper treated with whole-body hexane extract of a female. ently the case in a study of mate recognition in the longhorned beetle Megacyllene robiniae (Fo¨rster) (Ce- rambycinae) (Ginzel et al. 2003a). of this hypothesis, we applied one female equivalent Crude extracts of adult P. californicus contained 24 of solvent extract to a ground glass stopper and pre- different types of hydrocarbons (Table 1; Fig. 2). Pre- sented it to a male in the glass arena. The male re- liminary identiÞcation indicates that they are straight- peatedly attempted to copulate with the stopper (Fig. and branched chain alkanes with chain lengths rep- 3; i.e., a step 4 response), again conÞrming that rec- resenting the complete series from 24 to 29 carbons ognition cues were present in the extract, and that the (unpublished data). The cuticular hydrocarbon pro- mating response of males did not depend on tactile Þle is consistent in general structure with those of cues or behavioral signals of the female. Research to other cerambycid species (Kim et al. 1993; Ginzel et identify the contact pheromones of P. californicus is al. 2003a,b, 2006). There were considerable differ- currently underway. ences between the sexes in relative amounts of some compounds, with several compounds present in one Acknowledgments sex but not detected in the other (Table 1; Fig. 2). The most conspicuous difference between the sexes, how- We thank Joyce Ashcraft, Crystal Jones, Karen Barbour, ever, were greater relative amounts of compound 15 and Dan Cervantes for assistance with various aspects of this in females and compounds 17 and 18 in males (Table study and Jocelyn G. Millar for preliminary identiÞcation of cuticular hydrocarbons. This research was supported by a 1; Fig. 2). All compounds that apparently were sex Ͻ grant to J.D.B. from the Hop research Council, Busch Agri- speciÞc were relatively minor components ( 1% of cultural Resources, Inc., award K009607-ID6 from the West- the total). ern Region Integrated Pest Management Center, and a grant In other species of cerambycids, the most conspic- to L.M.H. from the Exotic/Invasive Pests and Diseases Re- uous differences between the sexes in cuticular hy- search Program, University of California, under USDAÐ drocarbon proÞles is that males have several com- CSREES Grant 2004-34439-14691. This is a publication of the pounds of greater chain length than those present in Idaho Agricultural Experiment Station. females (Ginzel et al. 2003a,b, 2006). All of the ap- parent male-speciÞc hydrocarbons of male P. califor- References Cited nicus, however, fall within the range of chain lengths present in females. Cuticular hydrocarbons of males Bishop, G. W., J. L. Blackmer, and C. R. Baird. 1984. Ob- that are absent or in lower abundance in females may servations on the biology of Prionus californicus Mots. on hops, Humulus lupulus L., in Idaho. J. Entomol. Soc. Br. serve as abstinons (male-produced compounds that Columbia 81: 20Ð24. prevent mating attempts between males; Schlein et al. Boyer, F.-D., C. Malosse, P. Zagatti, and J. Einhorn. 1997. 1981) and have been suggested to mediate mate rec- IdentiÞcation and synthesis of vesperal, the female sex ognition of another cerambycid species of the tribe pheromone of the longhorn beetle Vesperus xatarti. Bull. 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