Matthew D. Ginzel

Male-Produced Aggregation Pheromone of the Cerambycid Beetle Neoclytus acuminatus acuminatus

This is the first fully verified report of an aggregation pheromone produced by a cerambycid beetle species. Field bioassays with adultNeoclytus acuminatus acuminatus (F.) (Coleoptera: Cerambycidae) revealed that males produce a pheromone that attracts both sexes. Extracts of odors from males contained a single major male-specific compound, (2S,3S)-hexanediol. Field trials determined that both sexes were attracted by the racemic blend of (2S,3S)- and (2R,3R)-hexanediols and that activity was similar to enantiomerically enriched (2S,3S)-hexanediol (e.e. 80.2%). However, the blend of all four 2,3-hexanediol stereoisomers attracted few beetles, indicating inhibition by one or both of the (2R*,3S*)-stereoisomers. Females of the cerambycidCurius dentatus Newman were attracted to traps baited with the four component blend, suggesting that a male-produced sex pheromone for this species may contain (2R,3S)-hexanediol and/or (2S,3R)-hexanediol. The pheromone ofN. a. acuminatus, and presumed pheromone ofC. dentatus, bear structural similarities to those produced by males of six other species in the Cerambycinae (straight chains of 6, 8, or 10 carbons with hydroxyl or carbonyl groups at C2 and C3). It is likely that males of other species in this large subfamily produce pheromones that are variations on this structural motif.

Role of Contact Pheromones in Mate Recognition in Xylotrechus colonus

Adult male and female rustic borers, Xylotrechus colonus F. (Coleoptera: Cerambycidae), aggregate on cut logs and fallen trees that are the hosts of their larvae. Our studies show that male X. colonus actively search for females, and only respond to them after contacting them with their antennae. Stripping cuticular hydrocarbons from females with solvent rendered them unattractive to males, suggesting that males did not recognize females by mechanoreception alone. Reapplying solvent extract to washed females restored their attractiveness to males, confirming the role of cuticular hydrocarbons in mate recognition. Female cuticular hydrocarbon extracts contain n-pentacosane, 9-methylpentacosane, and 3-methylpentacosane, components that were either absent or present in very small amounts on males. We demonstrate that the contact pheromone is a blend of these three cuticular hydrocarbons.

ROLE OF HOST PLANT VOLATILES IN MATE LOCATION FOR THREE SPECIES OF LONGHORNED BEETLES

Stressed woody plants represent an ephemeral and unpredictable resource for larvae of some species of longhorned beetles (Coleoptera: Cerambycidae) because prime subcortical tissues are rapidly degraded by a guild of xylophagous competitors. Selection favors efficient mechanisms of host and mate location to expedite colonization of hosts by larvae. Based on previous research, we hypothesize that mate location in some species of the subfamily Cerambycinae involves three sequential behavioral stages: (1) both sexes are attracted to larval hosts by plant volatiles; (2) males attract females over shorter distances with pheromones; and (3) males recognize females by contact pheromones in their epicuticular wax layer. We already have evidence of second-stage and third-stage behaviors in three species in this subfamily whose xylophagous larvae feed in hardwood trees: Xylotrechus colonus, Megacyllene caryae, and Neoclytus mucronatus mucronatus. In this report, we evaluate the first behavioral stage of mate location behavior (i.e., independent response of both sexes to host plant volatiles) for the same three species. Supporting our hypothesis, both males and females responded to volatiles emanating from hickory logs in Y-tube olfactometer bioassays.

Contact Pheromones as Mate Recognition Cues of Four Species of Longhorned Beetles (Coleoptera: Cerambycidae)

We tested the hypothesis that contact phermones mediate mate recognition for four species of longhorned beetles, Neoclytus mucronatus mucronatus (F.), Megacyllene caryae (Gahan), Megacyllene robiniae (Forster), and Plectrodera scalator (F.). All tested males of all four species attempted to mate with females only after contacting them with their antennae. From 66.7 to 80% of tested males attempted to mate with hexane-extracted dead females treated with 0.1–1.0 female equivalents of conspecific female extracts, confirming that nonpolar compounds on the cuticle of females are essential for mate recognition in all four species. These findings are further evidence of the critical role of contact pheromones in mating systems of longhorned beetles.

(Z)-9-Pentacosene-contact sex pheromone of the locust borer, Megacyllene robiniae

Summary.Male locust borers, Megacyllene robiniae (Förster), responded to females only after contacting them with their antennae, indicating that mate recognition was mediated by a contact sex pheromone. GC-MS analyses of whole-body extracts of males and females determined that the profiles of compounds in the extracts were qualitatively similar, but differed considerably in the ratios of compounds between sexes. Biological activities of reconstructed blends of the most abundant straight-chain (nC23, nC24, nC25, nC26), methyl-branched (3me-C23, 3me-C25), and unsaturated (Z9:C23, Z9:C25, Z9:C27 compounds in extracts from females were assessed in arena bioassays, assessing four distinct steps in the mating behavior sequence of males (orientation, arrestment, body alignment, mounting and attempting to couple the genitalia). Males were unresponsive to freeze-killed, solventwashed females treated with blends of straight-chain and methyl-branched alkanes, but responded strongly to females treated with the blend of alkenes. Further trials determined that the complete sequence of mating behaviors, up to and including coupling the genitalia, was elicited by Z9:C25 alone. Z9:C25 comprised 16.4 ± 1.3% of the total hydrocarbons in whole-body hexane extracts of females and was co-dominant with two other hydrocarbons that were not active. In contrast, in solid phase microextraction (SPME) wipe samples from several areas of the cuticle, Z9:C25 appeared as the single dominant peak, comprising 34.6 − 37.8% of the sampled hydrocarbons. Our data indicate that Z9:C25 is a contact sex pheromone of M. robiniae, being the most abundant hydrocarbon on the surface of the cuticular wax layer of females where it is readily accessible to the antennae of males.

Fuscumol and fuscumol acetate are general attractants for many species of cerambycid beetles in the subfamily Lamiinae.

(E)‐6,10‐dimethyl‐5,9‐undecadien‐2‐ol (fuscumol) is an important component of male‐produced aggregation pheromones for several species of cerambycid beetles in the genus Tetropium (subfamily Aseminae/Spondylidinae). Here, we describe the experiments that tested the hypothesis that fuscumol and/or fuscumol acetate also are general attractants for species in the cerambycid subfamily Lamiinae. At field sites in northwestern Indiana and central Texas (USA), panel traps baited with fuscumol or its acetate captured 331 lamiine beetles, compared to 11 beetles captured in control traps. Three species were attracted to traps baited with fuscumol as a single component, whereas another four species were attracted to fuscumol acetate alone. Surprisingly, fuscumol acetate also attracted two species in the subfamily Cerambycinae: Xylotrechus colonus (Fabricius) (males of which produce a pheromone composed only of stereoisomers of 2,3‐hexanediol and 3‐hydroxyhexan‐2‐one), and Obrium maculatum (Olivier) (for which a pheromone has yet to be identified). In an independent field experiment in east‐central Illinois (USA), traps baited with fuscumol and/or its acetate captured 136 beetles of eight lamiine species, all but one species of which were also captured in the other experiment. Blending fuscumol and its acetate did not inhibit responses of species to either of the individual compounds, but synergized their activity for one species. Our results support the hypothesis that fuscumol and fuscumol acetate are widespread pheromone components or attractants for a variety of cerambycid species, especially lamiines in the tribe Acanthocinini.

(Z)-9-nonacosene-major component of the contact sex pheromone of the beetle Megacyllene caryae.

Male Megacyllene caryae (Gahan) (Coleoptera: Cerambycidae) respond to females only after touching them with their antennae, indicating that mate recognition is mediated by a contact sex pheromone. Gas chromatography-mass spectrometry analyses of whole-body solvent extracts of male and female M. caryae revealed substantial differences in hydrocarbon profiles, with nearly half of the compounds in the extracts from females being absent from those of males. Biological activities of fractions of crude extracts of females, and reconstructed blends of the most abundant straight-chain (nC27, nC28, nC29), methyl-branched (2Me-C26, 9Me-C29, 11, 13, 15Me-C29), and unsaturated (Z9:C29, Z13:C29, Z14:C29, Z13:C31, Z14:C31, Z15:C31) compounds in extracts of females were tested in arena bioassays, assessing four steps in the mating behavior sequence of males (orientation, arrestment, body alignment, mounting and attempting to couple the genitalia). Males showed limited response to dead females treated with fractions of the crude extract or blends of synthetic straight-chain and methyl-branched alkanes, but responded strongly to the blend of synthetic monoenes. Further trials determined that the complete sequence of mating behaviors, up to and including coupling the genitalia, was elicited by Z9:C29 alone. Z9:C29 is a homolog of the contact pheromone (Z9:C25) of the congener M. robiniae (Förster). Previous work with M. robiniae suggested that wipe sampling of cuticular hydrocarbons of females by solid phase microextraction yielded a more representative profile of components actually encountered by a male’s antennae, and so provided a more readily interpretable profile of potential semiochemicals present in the wax layer than does solvent extraction. We tested this hypothesis by comparing hydrocarbon profiles of female M. caryae by the two sampling methods. Z9:C29 was the only compound among the dominant hydrocarbons that was present in higher abundance in SPME than in solvent extracts (∼12% vs. ∼8%, respectively), supporting this hypothesis.

Evaluating western corn rootworm (Coleoptera: Chrysomelidae) emergence and root damage in a seed mix refuge.

ABSTRACT Resistance management is essential for maintaining the efficacy and long-term durability of transgenic corn engineered to control western corn rootworm (Diabrotica virgifera virgifera Le Conte). Theoretically, a refuge can be provided by growing susceptible (refuge) plants in either a separate section of the field adjacent to resistant (transgenic) plants, or as a seed mixture. We examined the effects of varying the structure of a 10 and 20% refuge between currently approved structured refuges (block or strip plantings), as well as deploying the refuge within a seed mix, on adult emergence timing and magnitude, root damage and yield. Our 2-yr field study used naturally occurring western corn rootworm populations and included seven treatments: 10 and 20% block refuge, 10 and 20% strip refuge, 10 and 20% seed mix refuge, and 100% refuge. Beetles emerging from refuge corn emerged more synchronously with those emerging from transgenic (Bacillus thuringiensis [Berliner] Bt-RW) corn in seed mix refuges when compared with block refuges. The proportion of beetles emerging from refuge plants was significantly greater in a block and strip refuge structure than in a seed mix refuge. More beetles emerged from Bt-RW corn plants when they were grown as part of a seed mix. We discuss the potential benefits and drawbacks of a seed mix refuge structure in light of these findings.

7-methylheptacosane is a major component of the contact sex pheromone of the cerambycid beetle Neoclytus acuminatus acuminatus

Abstract Male Neoclytus acuminatus acuminatus (F.) (Coleoptera: Cerambycidae) attempt to mate with females only after touching them with their antennae, suggesting that mate recognition is mediated by contact pheromones in the cuticular wax layer of females. Consistent with that hypothesis, males exhibit similar responses to dead females in laboratory bioassays, but not to solvent‐washed dead females with their cuticular hydrocarbons removed. The mating response of males is restored when solvent extracts are reapplied to carcasses of solvent‐washed females, indicating that the contact pheromone is present in solvent extracts. Solvent extracts of the female cuticle contain six methylalkanes that are not present in extracts of males, three of which (7Me‐C25, 7Me‐C27 and 9Me‐C27) constitute almost 40% of the total hydrocarbons. The bioactivity of these three compounds is tested by applying synthetic standards to solvent‐washed carcasses of females and presenting them to males. Standards are tested singly, pairwise and as the complete blend; freeze‐killed females serve as controls. Males attempt to couple with solvent‐washed female carcasses treated with 7Me‐C27 alone and in combination with 9Me‐C27 but only the complete blend elicits the same number of mounting and coupling attempts as does the control. These findings suggest that 7Me‐C27 (7‐methylheptacosane) is the major component of the contact sex pheromone of N. a. acuminatus and that 7Me‐C25 and 9Me‐C27 act as synergists.

First Contact Pheromone Identified for a Longhorned Beetle (Coleoptera: Cerambycidae) in the Subfamily Prioninae

Little is known of the reproductive behavior of longhorned beetles (Coleoptera: Cerambycidae) in the subfamily Prioninae. Mallodon dasystomus (Say), the hardwood stump borer, is a widely distributed prionine that is native to the southern U.S. Here, we explored the chemically-mediated mating behavior of M. dasystomus, and tested the hypothesis that males recognize females by a contact pheromone. In mating bioassays, all males tested attempted to mate with females only after contacting females with their antennae. Moreover, all males attempted to mate with solvent-washed dead females treated with as little as 0.15 ± 0.03 female equivalents of conspecific cuticular extracts, confirming that compounds on the cuticle of females are essential for mate recognition. Cuticular hydrocarbon profiles of females contained 13 compounds that were not present in profiles of males. Among the female-specific compounds, two co-dominant methyl-branched alkanes, 2-methylhexacosane (2Me-C26) and 2-methyloctacosane (2Me-C28), accounted for 17% of the total hydrocarbons. Our strategy for identifying the contact pheromone was to synthesize and test the bioactivity of female specific compounds, starting with the most abundant. In bioassays, males displayed mating behavior in response to synthetic 2Me-C26 and 2Me-C28 when tested individually. Furthermore, when these compounds were tested in combination, they elicited the full progression of mating behaviors, suggesting that 2Me-C26 and 2Me-C28 make up the contact pheromone. These findings are further evidence of the critical role of contact pheromones in mating systems of longhorned beetles.