4D Labs

Our objective was to identify the sex pheromone of Lymantria bantaizana (Lepidoptera: Lymantriidae) whose larvae feed exclusively on walnut, Juglans spp., in China, and Japan. Coupled gas chromatographic–electroantennographic detection (GC-EAD) analyses of pheromone gland extracts revealed a single EAD-active component. Retention index calculations of this compound on four GC columns suggested that it was a methyl-branched octadecadiene with conjugated double bonds. In GC-EAD analyses of 2-methyloctadecenes, (Z)-2-methyl-7-octadecene and (E)-2-methyl-7-octadecene elicited the strongest antennal responses, suggesting that the double bond positions were at C7 and C9. In comparative GC-EAD analyses of pheromone gland extract and stereoselectively synthesized isomers (E,E; E,Z; Z,E; Z,Z) of 2-methyl-7,9-octadecadiene, the (E,Z)- and (Z,E)-isomer had retention times identical to that of the candidate pheromone, but only the latter isomer elicited strong EAD activity. Results of field experiments in Japan substantiated that (7Z,9E)-2-methyl-7,9-octadecadiene is the L. bantaizana sex pheromone, a compound previously unknown in the Lepidoptera. Detection surveys in North America for exotic Eurasian forest defoliators could include traps baited with the L. bantaizana pheromone.

 Pheromone extract of the female orange wheat blossom midge, Sitodiplosis mosellana (Géhin) (SM) (Diptera: Cecidomyiidae), was analyzed by coupled gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometry (MS), employing fused silica columns coated with DB-5, DB-210, DB-23 or SP-1000. These analyses revealed a single, EAD-active candidate pheromone which was identified as 2,7-nonanediyl dibutyrate. In experiments in wheat fields in Saskatchewan, traps baited with (2S,7S)-2,7-nonanediyl dibutyrate attracted significant numbers of male SM. The presence of other stereoisomers did not adversely affect trap captures. Facile synthesis of stereoisomeric 2,7-nonanediyl dibutyrate will facilitate the development of pheromone-based monitoring or even control of SM populations.

(Z, Z)-6,9-Heneicosadien-11-one (Z6Z9-11-one-21Hy) was identified as the major sex pheromone component of the painted apple moth (PAM), Teia anartoides (Lepidoptera: Lymantriidae), on the basis of (1) comparative gas chromatographic-electroantennographic detection (GC-EAD) analyses, GC-mass spectrometry (MS), high-performance liquid chromatography (HPLC)-MS, and HPLC-UV/visible spectroscopy of pheromone gland extracts and authentic standards; (2) GC-EAD analyses of effluvia of calling females; and (3) wind tunnel and field trapping experiments with a synthetic standard. In field experiments in Australia, synthetic Z6Z9-11-one-21Hy as a single component attracted male moths. Wind tunnel experiments suggested that a 4-component blend consisting of Z6Z9-11-one-21Hy, (6Z,9R,10S)-cis-9,10-epoxy-heneicosene (Z6-9R10S-epo-21Hy), (E, E)-7,9-heneicosadien-6,11-dione (E7E9-6,11-dione-21Hy), and 6-hydroxy-(E, E)-7,9-heneicosadien-11-one (E7E9-6-ol-11-one-21Hy) (all present in pheromone gland extracts) might induce more males to orient toward, approach, and contact the source than did Z6Z9-11-one-21Hy as a single component. Additional experiments are needed to determine conclusively whether or not Z6-9R10S-epo-21Hy, E7E9-6,11-dione-21Hy, and E7E9-6-ol-11-one-21Hy might be minor sex pheromone components of PAM. Moreover, attractiveness of synthetic pheromone and virgin PAM females needs to be compared to determine whether synthetic pheromone could replace PAM females as trap baits in the program to monitor eradication of exotic PAM in New Zealand.

Three candidate sex pheromone components, (Z)6,(Z)9-, (Z)6,(E)8-, and (Z)6,(E)9-heneicosadien-11-one (Z6Z9, Z6E8, and Z6E9) were identified in pheromone gland extracts of female Douglas-fir tussock moths (DFTM), Orgyia pseudotsugata (McDunnough). Their occurrence in subnanogram quantities in extracts and structural conversion during analytical procedures and bioassays complicated chemical identifications. Complete identification required comparative analyses of stereoselectively synthesized and female-produced dienones by coupled gas chromatographic–electroantennographic detection (GC-EAD), high-performance liquid chromatography (HPLC) and coupled GC–mass spectrometry (MS). Determination of the pheromone component was contingent upon an experimental design that minimized structural rearrangement of dienones before and during the field test. In a 40-min field experiment, acetonitrile solutions of each of the above dienones were carried on Dry Ice to traps and were syringed onto cotton release devices below trap lids. In combination with the previously known sex pheromone component of DFTM, (Z)6-heneicosen-11-one (Z6), Z6E8 was the only synergistic dienone and the mixture was highly attractive. Because Z6 by itself attracts seven species of tussock moths (two sympatric with DFTM), a blend of Z6 and Z6E8 may impart specificity to DFTM pheromone communication. In commercial lures, this binary blend may facilitate species-specific, sensitive monitoring and efficacious control by mating disruption of this important forest defoliator.

Adults and juveniles of the common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), return to and aggregate in harborages after foraging for hosts. We tested the hypothesis that the aggregation is mediated, in part, by an airborne aggregation pheromone. Volatiles from experimental C. lectularius harborages were captured on Porapak Q, fractionated by liquid chromatography, and bioassayed in dual-choice, still-air olfactometer experiments. Of 14 compounds with >100 pg abundance in gas chromatography–mass spectrometry analyses of two bioactive fractions, 10 compounds [nonanal, decanal, (E)-2-hexenal, (E)-2-octenal, (2E,4E)-octadienal, benzaldehyde, (+)- and (−)-limonene, sulcatone, benzyl alcohol] proved to be essential components of the C. lectularius airborne aggregation pheromone.

(6Z,9Z,11S)-6,9-Heneicosadien-11-ol (Z6Z9-11S-ol-C21) and (6Z,9Z,11R)-6,9-heneicosadien-11-ol (Z6Z9-11R-ol-C21) were identified as major sex pheromone components of female tussock moths, Orgyia detritaGuérin-Méneville (Lepidoptera: Lymantriidae), on the basis of (1) analyses of pheromone gland extracts of female O. detrita by coupled gas chromatographic-electroantennographic detection (GC-EAD) and GC mass spectrometry, and (2) field trapping experiments with synthetic standards. Z6Z9-11S-ol-C21 and Z6Z9-11R-ol-C21 in combination, but not singly, attracted significant numbers of male moths. Racemic Z6Z9-11-ol-C21 was more attractive than the 1:3.5 (R:S) blend ratio found in pheromone gland extracts from female moths. Lower and higher homologues of Z6Z9-11-ol-C21 were also detected in GC-EAD recordings of pheromone extracts, and the racemic compounds enhanced attractiveness of Z6Z9-11-ol-C21 in field experiments. Because of trace amounts of these homologues in extracts, their enantiomeric composition could not be determined. This is the first report of secondary alcohols as pheromone components in the ditrysian (advanced) Lepidoptera.

Considering the vast Eurasian distribution of gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae), the many subspecies, and their presence in different lymantriid communities, we tested the hypothesis that L. dispar populations in eastern Asia employ one or more pheromone components in addition to the previously known single component pheromone (7R,8S)-cis-7,8-epoxy-2-methyloctadecane [= (+)-disparlure]. Coupled gas chromatographic–electroantennographic detection (GC–EAD) analyses of pheromone gland extracts of female L. dispar sensu lato (including both AGM and NAGM) on four GC columns (DB-5, DB-23, DB-210, and SP-1000) revealed a new trace component that eluted just before (DB-5; DB-210) or after (DB-23, SP-1000) disparlure, and elicited strong antennal responses. Isolation of this compound by high-performance liquid chromatography and hydrogenation produced disparlure, suggesting that the new component had the molecular skeleton of disparlure, with one or more double bonds. Of all possible monounsaturated cis-7,8-epoxy-2-methyloctadecenes, only cis-7,8-epoxy-2-methyloctadec-17-ene co-chro- matographed with the insect-produced compound on all GC columns and elicited comparable antennal responses. In field experiments in Honshu (Japan) with enantioselectively synthesized compounds, (7R,8S)-cis-7,8-epoxy-2- methyloctadec-17-ene (7R8S-epo-2me-17-ene-18Hy) was weakly attractive to male L. dispar, but was less effective as a trap bait than (+)-disparlure, and failed to enhance attractiveness of (+)-disparlure when tested in blends. The antipode, (7S,8R)-cis-7,8-epoxy-2-methyloctadec-17-ene, was not attractive, and when added to (+)-disparlure and/or 7R8S-epo-2me-17-ene-18Hy reduced attractiveness. Thus, the biological role of 7R8S-epo-2me-17-ene-18Hy remains unclear. It may enhance pheromone attractiveness or specificity in other L. dispar populations within their vast Eurasian distribution.

The Indian gypsy moth, Lymantria obfuscata (Lepidoptera: Lymantriidae), has been recognized as a distinct species since 1865 but closely resembles a diminutive form of gypsy moth, Lymantria dispar. We tested the hypothesis that the sex pheromones of L. obfuscata and L. dispar are similar. In laboratory mate acceptance studies, very few male L. dispar made copulatory attempts when paired with female L. obfuscata, suggesting that female L. obfuscata emit one or more pheromone components antagonistic to male L. dispar. In coupled gas chromatographic–electroantennographic detection (GC–EAD) analyses of pheromone gland extract of female L. obfuscata, (Z)-2-methyloctadec-7-ene (2Me-7Z-18Hy) and (7R,8S)-cis-7,8-epoxy-2-methyloctadecane [(+)-disparlure] were most abundant and elicited the strongest responses from male L. obfuscata antennae. In field experiments near Solan (Himachal Pradesh, India), 2Me-7Z-18Hy and (+)-disparlure in combination attracted more male L. obfuscata than did either component alone. This two-component sex pheromone contrasts with the single-component sex pheromone [(+)-disparlure] of L. dispar. The contrasting composition of the lymantriid communities inhabited by L. obfuscata and L. dispar may explain why 2Me-7Z-18Hy is a pheromone component in L. obfuscata and a pheromone antagonist in L. dispar and why (−)-disparlure reduces pheromonal attraction of male L. dispar but not male L. obfuscata.

We determined that location of host (Cydia pomonella) eggs by Ascogaster quadridentata is mediated by kairomones, investigated potential sources of the kairomones and identified a blend of kairomones from the source that was attractive to A. quadridentata. In Y-tube olfactometer bioassays, female A. quadridentata were attracted to Porapak Q-collected volatiles from female C. pomonella scales and eggs, but not to C. pomonella sex pheromone. Scales of C. pomonella were also attractive to male A. quadridentata. Coupled gas chromatographic–electroantennographic detection analysis of scale volatile extracts revealed numerous compounds that elicited responses from male or female A. quadridentata antennae, including heptanal, octanal, nonanal, decanal, undecan-2-one, dodecanal, pentadecan-2-one, (Z)-6-pentadecen-2-one, (Z)-9-hexadecenal, (Z)-6-heptadecen-2-one, and 3,7,11-trimethyl-2E,6E,10-dodecatrien-1-ol acetate. A synthetic blend of these compounds at quantities and ratios equivalent to Porapak Q scale volatile extract was attractive to female A. quadridentata in a Y-tube olfactometer bioassay.

Gas chromatographic–electroantennographic detection (GC-EAD) analyses of pheromone gland extracts of female nettle caterpillars, Setora nitens, revealed four compounds that consistently elicited responses from male moth antennae. Retention indices on three fused silia columns (DB-5, DB-23, and DB-210) of two EAD-active compounds were almost identical to those of (E)-9-dodecenal (E9–12 : Ald) and (E)-9,11-dodecadienal (E9,11–12 : Ald), two pheromone components previously identified in congeneric Setothosea asigna. However, comparative GC, GC-EAD, and GC-mass spectrometry of extracted S. nitens compounds and authentic standards revealed that the candidate pheromone components were (Z)-9-dodecenal (Z9–12 : Ald) and (Z)-9,11-dodecadienal (Z9,11–12 : Ald). The two other EAD-active compounds in pheromone gland extracts proved to be the corresponding alcohols to these aldehydes. In field-trapping experiments in Tawau, Malaysia, synthetic Z9–12 : Ald and Z9,11–12 : Ald at a 1 : 1 ratio, but not singly, attracted male S. nitens. Attractiveness of these two aldehydes could not be enhanced through the addition of their corresponding alcohols. Whether these differences in pheromone biology and chemistry between S. nitens and S. asigna are sufficient to prevent cross-attraction of heterospecific males or whether nonpheromonal mechanisms are required to maintain reproductive isolation is currently being studied.

In a recent study, evidence was presented that females of the aphidophagous midge Aphidoletes aphidimyza (Rondi) (Diptera: Cecidomyiidae) release a sex pheromone to attract mates. Our objectives were to identify and bioassay the pheromone. Coupled gas chromatographic–electroantenno- graphicDetection (GC-EAD) analyses of untreated and hydrogenated pheromone extract on three fused-silica columns (DB-5, DB-23, DB-210) revealed a single compound that elicited responses from male antennae. Retention index calculations of this candidate pheromone (CP) suggested that it was a di-acetate. Considering that most of the presently identified cecidomyiid pheromones consist of a 13-carbon chain with (at least) one acetate group in C2, we synthesized 2,6-, 2,7-, 2,8-, 2,9-, 2,10-, 2,11-, and 2,12-diacetoxytridecane. In GC analyses of these compounds, only 2,7-diacetoxytridecane cochomatographed with CP on all columns. In laboratory two-choice experiments with stereospecifically synthesized stereoisomers, only (2R,7S)-diacetoxytridecane elicited significant anemotatic responses by male A. aphidimyza. In trapping experiments in greenhouse compartments, only traps baited with (2R,7S)-diacetoxytridecane captured significant numbers of male A. aphidimyza, clearly revealing the absolute configuration of the pheromone. Failure of the stereoisomeric mixture (containing all four stereoisomers including the pheromone) to attract males is due to inhibitory characteristics of the (2R,7R)- and (2S,7R)-stereoisomers. The pheromone of zoophagous A. aphidimyza resembles those from phytophagous cecidomyiid midges, suggesting a common, diet-independent pathway for pheromone biosyntheses.

Our objectives were to identify and field test the sex pheromone of female Douglas-fir cone gall midge, Contarinia oregonensis (Diptera: Cecidomyiidae). Coupled gas chromatographic–electroantennographic detection (GC-EAD) analyses of pheromone extract revealed a single compound (A) that elicited responses from male antennae. Hydrogenation of pheromone extract, followed by renewed GC-EAD analysis, revealed a new EAD-active compound with chromatographic characteristics identical to those of tridecan-2-yl acetate on five fused silica columns (DB-5, DB-210, DB-23, SP-1000, and Cyclodex-B). Syntheses, chromatography, and retention index calculations of all possible tridecen-2-yl acetates suggested that the candidate pheromone A was a tridecadien-2-yl acetate with nonconjugated double bonds. Synthetic candidate pheromone component (Z,Z)-4,7-tridecadien-2-yl acetate (Z4Z7) cochromatographed with A on all analytical columns and elicited comparable antennal activity. In GC-EAD analyses that separated the enantiomers (Z,Z)-4,7-tridecadien-(S)-2-yl acetate (2S-Z4Z7) and (Z,Z)-4,7-tridecadien-(R)-2-yl acetate (2R-Z4Z7) with baseline resolution, only 2S-Z4Z7 as a component in a racemic standard or in pheromone extract elicited antennal responses. In Douglas-fir seed orchards, sticky traps baited with 2S-Z4Z7 captured male C. oregonensis, whereas 2R-Z4Z7 was behaviorally benign. Comparable catches of males in traps baited with racemic Z4Z7 (50 μg) or virgin female C. oregonensis suggested that synthetic pheromone baits could be developed for monitoring C. oregonensis populations in commercial Douglas-fir seed orchards.

6Z ,9Z ,11S)-6,9-Heneicosadien-11-ol (Z 6Z 9-11S-ol-C21) and (6Z ,9Z ,11R)-6,9-heneicosadien-11-ol (Z 6Z 9-11R-ol-C21) were identified as major sex pheromone components of female tussock moths, Orgyia detrita Guérin-Méneville (Lepidoptera: Lymantriidae), on the basis of (1) analyses of pheromone gland extracts of female O. detrita by coupled gas chromatographicelectroantennographic detection (GC-EAD) and GC mass spectrometry, and (2) field trapping experiments with synthetic standards. Z 6Z 9-11S-ol-C21 and Z 6Z 9-11R-ol-C21 in combination, but not singly, attracted significant numbers of male moths. Racemic Z 6Z 9-11-ol-C21 was more attractive than the 1:3.5 (R:S) blend ratio found in pheromone gland extracts from female moths. Lower and higher homologues of Z 6Z 9-11-ol-C21 were also detected in GC-EAD recordings of pheromone extracts, and the racemic compounds enhanced attractiveness of Z 6Z 9-11-ol-C21 in field experiments. Because of trace amounts of these homologues in extracts, their enantiomeric composition could not be determined. This is the first report of secondary alcohols as pheromone components in the ditrysian (advanced) Lepidoptera.

(E)-11,13-Tetradecadienal (E11,13–14:Ald) is the major sex pheromone component of the eastern blackheaded budworm (EBB),Acleris variana (Fern.). The compound was identified in female pheromone gland extracts by coupled gas chromatographic-electroantennographic detection (GC-EAD), coupled GC-mass spectrometry in selected ion monitoring mode, and retention index calculations of candidate pheromone components.E11,13–14:Ald alone as trap bait was very attractive to male EBB. Addition of the corresponding diene alcohol or acetate or both did not enhance attraction. (Z)-11,13-Tetradecadienal in binary combination with (E)-11,13–14:Ald neither enhanced nor reduced trap catches. Increasing the amounts of pheromone from 0.01 to 10 µg increased trap catches, but increase of pheromone quantity above 100 µg proportionately reduced attraction. Stabilization of slowly polymerizingE11,13–14:Ald and development of a sustained, adequate release rate is required for pheromone-based monitoring of EBB populations.

Pheromonal communication of adult peach twig borers, Anarsia lineatella Zeller (Lepidoptera: Gelechiidae), was reinvestigated based on recent findings that virgin female-baited traps were more attractive to mate-seeking males than a two-component synthetic sex pheromone consisting of (E)-5-decen-1-yl acetate (1000 μg) and (E)-5-decen-1-ol (100 μg), suggesting that females use additional pheromone components. Hypothesizing that these additional components may be released from body parts other than abdominal sex pheromone glands, we extracted female body scales and analyzed aliquots by coupled gas chromatographic–electroantennographic detection (GC-EAD) and GC–mass spectrometry. Eight straight-chain and four methylated aliphatic hydrocarbons, as well as two acetates, all elicited responses from excised male antennae. In laboratory experiments with synthetic candidate pheromone components, a combination of octadecyl acetate, (R)-11-methyltricosane, and (S)-11-methyltricosane in the presence of gland-derived sex pheromone components were shown to elicit contact of female decoys by males. However, body pheromone components did not enhance attractiveness of sex pheromone components in field trapping experiments, suggesting that they are effective only at close range and that other stimuli are responsible for superior attractiveness of female-baited traps.

This study was undertaken to identify sex pheromone components of nettle caterpillars Darna trima and Darna bradleyi (Lepidoptera: Limacodidae) whose larvae defoliate oil palm, Elaeis guineensis, in southeast Asia. Coupled gas chromatographic–electroantennographic detection (GCEAD) analyses of pheromone gland extracts revealed two antennally active compounds produced by female D. trima and two by female D. bradleyi. Molecular structures of these candidate pheromone components were identified by electron-impact and chemical-ionization mass spectrometry; retention-index calculations on DB-5, DB-23, and DB-210 columns; microanalytical treatments, as well as syntheses of "auxilliary" compounds that facilitated identification of the compounds. The compounds from D. trima were 2-methylbutyl (E)-7,9-decadienoate (A) and (E)-2-hexenyl (E)-7,9decadienoate (B); from D. bradleyi we identified methyl (E)-7,9-decadienoate (C), and isobutyl (E)-7,9-decadienoate (D). In field experiments in Malaysia, (S)-2-methylbutyl (E)-7,9-decadienoate (SA) in combination with B proved to be essential and synergistic pheromone components for attraction of male D. trima. (R)-2-Methylbutyl (E)-7,9-decadienoate (RA) had no behavioral activity. Compound D singly attracted male D. bradleyi, but addition of C to D at a 1 : 10 ratio significantly enhanced attractiveness of the bait. Synthetic pheromone blends were more effective trap baits than unmated female moths and could be developed for monitoring populations of D. trima and D. bradleyi in Asian oil palm plantations.