Pheromonal Activity of Compounds Identified from Male <Emphasis Type="Italic">Phyllotreta cruciferae</Emphasis>: Field Tests of Racemic Mixtures,Pure Enantiomers,and Combinations with Allyl Isothiocyanate

Four himachalene sesquiterpenes and (+)-γ-cadinene, previously identified as possible pheromone components from males of a North American population of Phyllotreta cruciferae Goeze (Coleoptera, Chrysomelidae), were tested for attractiveness in field trapping experiments in Hungary. A mixture of the four synthetic racemic himachalene derivatives and (+)-γ-cadinene from a botanical source was slightly attractive to beetles, but much more attractive when blended with the known host-plant-derived attractant allyl isothiocyanate. This result was consistent with a previous study in North America. In tests with optically pure synthetic compounds, a blend of the same himachalene enantiomers found from male beetles was equivalent to the corresponding blend of racemic compounds, whereas a blend of the opposite enantiomers was not active. Through subtraction tests, it was found that the single compound, (6R,7S)-2,2,6,10-tetramethylbicyclo[5.4.0.]undeca-9,11-diene [compound (+)-A in this study], was as active as the whole mixture, suggesting that this compound is the key pheromone component of the European population of P. cruciferae. During field trials, several congeneric species, including P. vittula, P. nemorum, P. nodicornis, and P. ochripes, also were caught, suggesting that the same compound(s) may be relatively widespread as pheromone components in this genus.     

Male-Produced Aggregation Pheromones of the Cerambycid Beetles <Emphasis Type="Italic">Xylotrechus colonus</Emphasis> and <Emphasis Type="Italic">Sarosesthes fulminans</Emphasis>

Adults of both sexes of the cerambycid beetles Xylotrechus colonus (F.) and Sarosesthes fulminans (F.) were attracted to odors produced by male conspecifics in olfactometer bioassays. Analyses of headspace volatiles from adults revealed that male X. colonus produced a blend of (R)- and (S)-3-hydroxyhexan-2-one and (2 S,3 S)- and (2R,3R)-2,3-hexanediol, whereas male S. fulminans produced (R)-3-hydroxyhexan-2-one and (2 S,3R)-2,3-hexanediol. All of these compounds were absent in the headspace of females. Two field bioassays were conducted to confirm the biological activity of the synthesized pheromones: (1) enantiomerically enriched pheromone components were tested singly and in species-specific blends and (2) four-component mixture of racemic 3-hydroxyhexan-2-one plus racemic 2-hydroxyhexan-3-one and the four-component blend of the stereoisomers of 2,3-hexanediols were tested separately and as a combined eight-component blend. In these experiments, adult male and female X. colonus were captured in greatest numbers in traps baited with the reconstructed blend of components produced by males, although significant numbers were also captured in traps baited with (R)-3-hydroxyhexan-2-one alone or in blends with other compounds. Too few adult S. fulminans were captured for a statistical comparison among treatments, but all were caught in traps baited with lures containing (R)-3-hydroxyhexan-2-one. In addition to these two species, adults of two other species of cerambycid beetles, for which pheromones had previously been identified, were caught: Neoclytus a. acuminatus (F.) and its congener Neoclytus m. mucronatus (F.). Cross-attraction of beetles to pheromone blends of other species, and to individual pheromone components that are shared by two or more sympatric species, may facilitate location of larval hosts by species that compete for the same host species.     

Sex Pheromone of the Dogwood Borer, <Emphasis Type="Italic">Synanthedon scitula</Emphasis>

The sex pheromone of female dogwood borers (DWB) Synanthedon scitula (Harris) (Lepidoptera: Sesiidae) was determined to be an 88:6:6 ternary blend of (Z,Z)-3,13-octadecadienyl acetate (Z,Z-3,13-ODDA), (E,Z)-2,13-octadecadienyl acetate (E,Z-2,13-ODDA), and (Z,E)-3,13-octadecadienyl acetate (Z,E-3,13-ODDA) by gas chromatography–electroantennographic detection (GC–EAD) and gas chromatography–mass spectrometry (GC–MS). The major sex pheromone component, Z,Z-3,13-ODDA, was attractive as a single component. A blend of Z,Z-3,13-ODDA with 1–3% of E,Z-2,13-ODDA (binary blend) was more attractive than the single component. A third component, Z,E-3,13-ODDA, was sometimes observed in GC–EAD analyses, and enhanced attraction to the binary blend in some field bioassays. Lures containing 1 mg of binary and ternary blends attracted 18 and 28 times more male DWB moths, respectively, than caged virgin females in field trials. Attraction was strongly antagonized by addition of as little as 0.5% of E,Z-3,13-octadecadienyl acetate (E,Z-3,13-ODDA). In a period of 12 wk in 2004, more than 60,000 males were captured in sticky traps baited with synthetic pheromone blends in six apple orchards in Virginia, West Virginia, and North Carolina. Lure longevity trials showed that ∼76% of the pheromone remained in rubber septum lures after 12 wk in the field.     

A Male-produced Aggregation Pheromone Blend Consisting of Alkanediols,Terpenoids, and an Aromatic Alcohol from the Cerambycid Beetle <Emphasis Type="Italic">Megacyllene caryae</Emphasis>

Bioassays conducted with a Y-tube olfactometer provided evidence that both sexes of the cerambycid beetle Megacyllene caryae (Gahan) were attracted to odor produced by males. Odor collected from male M. caryae contained eight male-specific compounds: a 10:1 blend of (2S,3R)- and (2R,3S)-2,3-hexanediols (representing 3.2 ± 1.3% of the total male-specific compounds), (S)-(−)-limonene (3.1 ± 1.7%), 2-phenylethanol (8.0 ± 2.4%), (−)-α-terpineol (10.0 ± 2.8%), nerol (2.1 ± 1.5%), neral (63.3 ± 7.3%), and geranial (8.8 ± 2.4%). Initial field bioassays determined that none of these compounds was attractive as a single component. Further field trials that used a subtractive bioassay strategy determined that both sexes were attracted to the complete blend of synthetic components, but the elimination of any one component resulted in a decline in trap captures. Blends that were missing (2S,3R)-2,3-hexanediol, (2R,3S)-2,3-hexanediol, or citral (a 1:1 mixture of neral and geranial) attracted no more beetles than did controls. A pheromone blend of this complexity, composed of alkanediols, terpenoids, and aromatic alcohols, is unprecedented for cerambycid species.     

Sex Pheromone of <Emphasis Type="Italic">Elater ferrugineus</Emphasis> L. (Coleoptera: Elateridae)

The rare European click beetle, Elater ferrugineus L., develops exclusively in old, hollow deciduous trees. As a result of massive habitat loss caused by modern forestry, it is threatened throughout its entire distribution range and regarded as an indicator species for undamaged natural forests. As E. ferrugineus lives cryptically and its populations are frequently overlooked, we investigated its sex pheromone to develop a reliable detection tool. Pheromone gland extracts of single female E. ferrugineus were examined by using gas chromatography–mass spectrometry (GC–MS). All samples contained 7-methyloctyl 5-methylhexanoate, 7-methyloctyl octanoate, 7-methyloctyl 7-methyloctanoate, and 7-methyloctyl (Z)-4-decenoate in a ratio of approximately 1:1:3:3. Structures of all four esters, which have not been reported as pheromone compounds before, were confirmed by synthesis. A blend of the components was tested in the field and proved to be attractive for E. ferrugineus males, which were found to swarm exclusively during the day. This blend provides a noninvasive and effective monitoring method for this cryptic species, promising future collection records of E. ferrugineus in regions where it exists below the limit of detection by conventional collecting methods.     

Sex Pheromones and Reproductive Isolation of Three Species in Genus <Emphasis Type="Italic">Adoxophyes</Emphasis>

We tested differences in female pheromone production and male response in three species of the genus Adoxophyes in Korea. Females of all three species produced mixtures of (Z)-9-tetradecenyl acetate (Z9–14:OAc) and (Z)-11-tetradecenyl acetate (Z11–14:OAc) as major components but in quite different ratios. The ratio of Z9–14:OAc and Z11–14:OAc in pheromone gland extracts was estimated to be ca. 100:200 for Adoxophyes honmai, 100:25 for Adoxophyes orana, and 100:4,000 for Adoxophyes sp. Field tests showed that males of each species were preferentially attracted to the two-component blends of Z9–14:OAc and Z11–14:OAc mimicking the blends found in pheromone gland extracts of conspecific females. The effects of minor components identified in gland extracts on trap catches varied with species. Addition of 10-methyldodecyl acetate (10me-12:OAc) or (E)-11-tetradecenyl acetate (E11–14:OAc) to the binary blend of Z9–14:OAc and Z11–14:OAc significantly increased captures of A. honmai males, whereas E11–14:OAc exhibited a strongly antagonistic effect on catches of Adoxophyes sp. males. Moreover, (Z)-9-tetradecen-1-ol (Z9–14:OH) or (Z)-11-tetradecen-1-ol (Z11–14:OH) added to the binary blends increased attraction of male A. orana but not A. honmai and Adoxophyes sp. males, suggesting that these minor components, in addition to the relative ratios of the two major components, play an important role in reproductive isolation between Adoxophyes species in the southern and midwestern Korea where these species occur sympatrically.     

Aggregation Pheromone of the Qinghai Spruce Bark Beetle, <Emphasis Type="Italic">Ips nitidus</Emphasis> Eggers

Volatiles from hindgut extracts of males of the Qinghai spruce bark beetle, Ips nitidus, from different attack phases (phase 1: unpaired males and phases 2–4: males joined with one to three females) and hindgut extracts of mated females were analyzed by gas chromatography–mass spectrometry (GC–MS)/flame ionization detection (FID) with both polar and enantioselective columns. The GC–MS/FID analyses demonstrated that unpaired males from attack phase 1 (nuptial chamber constructed) produced 2-methyl-3-buten-2-ol, approx. 74%-(−)-ipsdienol, and (−)-cis-verbenol as major hindgut components, and (−)-trans-verbenol, (−)-ipsenol, (−)-verbenone, myrtenol, and 2-phenylethanol as minor or trace components. The quantities of 2-methyl-3-buten-2-ol and especially ipsdienol decreased after mating during phases 2–4, whereas the quantities of (−)-cis- and (−)-trans-verbenol did not change. In contrast, the quantity of (−)-ipsenol seemed to increase as mating activity progressed. After mating with three females (harem size = 3; phase 4), only trace to small amounts of male-specific compounds were detected from I. nitidus male hindguts. Chemical analysis of the hindgut extracts of mated females showed only trace amounts of semiochemicals. A field-trapping bioassay in Qinghai, China showed that the four-component “full blend” containing the three major components, 2-methyl-3-buten-2-ol, (±)-ipsdienol, and (−)-cis-verbenol, plus a minor component, (−)-trans-verbenol, caught significantly more I. nitidus (♂/♀ = 1:2.2) than did the unbaited control and two binary blends. The replacement of (±)-ipsdienol with nearly enantiomerically pure (−)-ipsdienol in the “full blend” significantly reduced trap catches, which suggests that both enantiomers are needed for attraction. On the other hand, removal of (−)-trans-verbenol from the active “full blend” had no significant effect on trap catches. Our results suggest that the three major components, 2-methyl-3-buten-2-ol, 74%-(−)-ipsdienol, and (−)-cis-verbenol (at 7:2:1), produced by unpaired fed males, are likely the aggregation pheromone components of I. nitidus, thus representing the first characterization of an aggregation pheromone system of a bark beetle native solely to China.     

Behaviorally Active Green Leaf Volatiles for Monitoring the Leaf Beetle, Diorhabda elongata, a Biocontrol Agent of Saltcedar, Tamarix spp.

Biological activity and chemistry of host plant volatiles were investigated for Diorhabda elongata, Brullé (Coleoptera: Chrysomelidae), a biological control agent for the invasive tree, saltcedar (Tamarix spp., Tamaricaceae). Gas chromatographic–electroantennographic detection (GC-EAD) analysis of volatiles collected from adult D. elongata feeding on saltcedar foliage or from saltcedar foliage alone showed 15 antennally active compounds. These compounds were more abundant in collections from beetle-infested foliage. Antennally active compounds were identified by GC–mass spectrometry (MS) and confirmed with authentic standards. The emissions of the most abundant GC-EAD-active compounds, green leaf volatiles (GLV), were quantitated by GC-MS. A blend of four GLV compounds, mimicking the natural blend ratio, was highly attractive to male and female D. elongata in the field, and a combination of GLV and male-produced aggregation pheromone attracted significantly greater numbers of D. elongata than did either bait alone. A preliminary experiment with a blend of seven additional GC-EAD-active saltcedar volatiles did not show any behavioral activity. The combination of the pheromone and the green leaf odor blend could be a useful attractant in detecting the presence of the biocontrol agent, D. elongata, in stands of saltcedar newly colonized by the beetle.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Disruption of Secondary Attraction of the Spruce Beetle, Dendroctonus rufipennis, by Pheromones of Two Sympatric Species

Capture of spruce beetles, Dendroctonus rufipennis, in multiple-funnel traps baited with frontalin and -pinene was reduced by up to 42% in the presence of synthetic (+)-exo- and (+)-endo-brevicomin, aggregation pheromones of the sympatric species Dryocoeles affaber. (±)-endo-Brevicomin was inhibitory to spruce beetles in two experiments and (±)-exo-brevicomin was inhibitory in one experiment, reducing spruce beetle trap catches by up to 87% and 75%, respectively. Spruce beetle trap catches were also reduced by 85% by (±)- or (+)-ipsdienol, but not by (–)-ipsdienol. Ips tridens, a second sympatric species, produces both enantiomers of ipsdienol in its pheromone blend. Responses by D. affaber to its own pheromone were significantly enhanced by addition of the spruce beetle lure. Enantiospecific pheromones of secondary competing species, or less costly racemic substitutes, may be useful for managing spruce beetles using competitive displacement or exclusion. Baiting susceptible hosts with pheromones of secondary species may enhance attack by secondary species, while partially repelling spruce beetles.     

Identification of Sex Pheromone Components of the Hessian Fly, <Emphasis Type="Italic">Mayetiola destructor</Emphasis>

Coupled gas chromatographic (GC)–electroantennographic detection (EAD) analyses of ovipositor extract of calling Hessian fly, Mayetiola destructor, females revealed that seven compounds elicited responses from male antennae. Four of the compounds—(2S)-tridec-2-yl acetate, (2S,10Z)-10-tridecen-2-yl acetate, (2S,10E)-10-tridecen-2-yl acetate, and (2S,10E)-10-tridecen-2-ol—were identified previously in female extracts. Two new EAD-active compounds, (2S,8Z,10E)-8,10-tridecadien-2-yl acetate and (2S,8E,10E)-8,10-tridecadien-2-yl acetate, were identified by GC–mass spectroscopy (MS) and the use of synthetic reference samples. In a Y-tube bioassay, a five-component blend (1 ng (2S)-tridec-2-yl acetate, 10 ng (2S,10E)-10-tridecen-2-yl acetate, 1 ng (2S,10E)-10-tridecen-2-ol, 1 ng (2S,8Z,10E)-8,10-tridecadien-2-yl acetate, and 1 ng (2S,8E,10E)-8,10-tridecadien-2-yl acetate) was as attractive to male Hessian flies as a similar amount of female extract (with respect to the main compound, (2S,10E)-10-tridecen-2-yl acetate). The five-component blend was more attractive to male flies than a three-component blend lacking the two dienes. Furthermore, the five-component blend was more attractive than a blend with the same compounds but that contained one tenth the concentration of (2S,8E,10E)-8,10-tridecadien-2-yl acetate (more accurately mimicking the ratios found in female extract). This suggests that the ratios emitted by females might deviate from those in gland extracts. In a field-trapping experiment, the five-component blend applied to polyethylene cap dispensers in a 100:10 μg ratio between the main component and each of the other blend components attracted a significant number of male Hessian flies. Also, a small-plot field test demonstrated the attractiveness of the five-component blend to male Hessian flies and suggests that this pheromone blend may be useful for monitoring and predicting Hessian fly outbreaks in agricultural systems.     

Aphid and Plant Volatiles Induce Oviposition in an Aphidophagous Hoverfly

Episyrphus balteatus DeGeer (Diptera, Syrphidae) is an abundant and efficient aphid-specific predator. We tested the electroantennographic (EAG) response of this syrphid fly to the common aphid alarm pheromone, (E)-β-farnesene (EβF), and to several plant volatiles, including terpenoids (mono- and sesquiterpenes) and green leaf volatiles (C6 and C9 alcohols and aldehydes). Monoterpenes evoked significant EAG responses, whereas sesquiterpenes were inactive, except for the aphid alarm pheromone (EβF). The most pronounced antennal responses were elicited by six and nine carbon green leaf alcohols and aldehydes [i.e., (Z)-3-hexenol, (E)-2-hexenol, (E)-2-hexenal, and hexanal]. To investigate the behavioral activity of some of these EAG-active compounds, E. balteatus females were exposed to R-(+)-limonene (monoterpene), (Z)-3-hexenol (green leaf alcohol), and EβF (sesquiterpene, common aphid alarm pheromone). A single E. balteatus gravid female was exposed for 10 min to an aphid-free Vicia faba plant that was co-located with a semiochemical dispenser. Without additional semiochemical, hoverfly females were not attracted to this plant, and no oviposition was observed. The monoterpene R-(+)-limonene did not affect the females’ foraging behavior, whereas (Z)-3-hexenol and EβF increased the time of flight and acceptance of the host plant. Moreover, these two chemicals induced oviposition on aphid-free plants, suggesting that selection of the oviposition site by predatory hoverflies relies on the perception of a volatile blend composed of prey pheromone and typical plant green leaf volatiles.     

Genetic Control of the Enantiomeric Composition of Ipsdienol in the Pine Engraver, Ips pini

The genetic nature of pheromone variation within species has rarely been studied, and never for male-produced long-range pheromones. Males from western North American populations of Ips pini produce predominantly (R)-(−)-ipsdienol, whereas those from eastern North American populations produce higher proportions of (S)-(+)-ipsdienol. From a population in the hybrid zone, we divergently selected lines for the opposing pheromonal types and then created F1, F2, and backcross lines. We formed additional F1, F2, and backcross lines, first by using populations with low (+)-ipsdienol enantiomeric ratios near to and distant from the hybrid zone, and then by using populations with high (+)-ipsdienol enantiomeric ratios near to and distant from the hybrid zone. Three types of analysis were employed: (1) line means analysis; (2) Mendelian analysis of assigned high and low (+)-ipsdienol enantiomeric ratio phenotypes when applicable; and (3) Castle–Wright estimation of the number of effective factors. Dominance at one autosomal locus explains much of the variation in ipsdienol blend between the divergently selected lines. Thus, as in all previously studied female long-range pheromone systems, a major genetic element is implicated. The populations with low (+)-ipsdienol enantiomeric ratios near and distant to the hybrid zone differ negligibly for this trait. We confirmed previous studies showing slightly higher ratios of (+)-ipsdienol at the hybrid zone than in a distant eastern population and reveal a genetic basis for this difference.     

Pheromone Components from Body Scales of Female <Emphasis Type="Italic">Anarsia lineatella</Emphasis> Induce Contacts by Conspecific Males

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.     

A Male-produced Sex Pheromone from the Neotropical Redbanded Stink Bug, <Emphasis Type="Italic">Piezodorus guildinii</Emphasis> (W.)

The male-produced sex pheromone of the neotropical redbanded stink bug, Piezodorus guildinii (W.) (Hemiptera: Pentatomidae), a serious pest of soybean in Central and South America, was identified as the sesquiterpene hydrocarbon β-sesquiphellandrene by coupled gas chromatography–mass spectrometry and laboratory bioassays. The identity was confirmed by comparison with an authentic standard, and its absolute configuration was determined to be (7R)-(+)-β-sesquiphellandrene. The mean rate of release was approximately 40 ng d⁻¹ male⁻¹. The compound was found only in extracts from sexually mature males. Laboratory olfactometer bioassays showed that 200 ng of the synthetic compound applied to a filter paper strip was as attractive to female bugs as odors from five 15-d-old live males, an airborne extract from males, or the fraction of the extract that contained (7R)-(+)-β-sesquiphellandrene.     

Enantiomeric Specificity in a Pheromone–Kairomone System of Two Threatened Saproxylic Beetles, Osmoderma eremita and Elater ferrugineus

The scarab beetle Osmoderma eremita and its larval predator, the click beetle Elater ferrugineus, are threatened saproxylic beetles regarded as indicators of the species-richness of insect fauna of hollow deciduous trees. Male O. eremita produce the pheromone (R)-(+)-γ-decalactone to attract conspecific females, and this compound is also utilized by E. ferrugineus as a kairomone, presumably for detection of tree hollows containing prey. We have investigated enantiomeric specificity to γ-decalactone in this pheromone–kairomone system by electrophysiological and field trapping experiments. In single-sensillum recordings from male and female O. eremita, which used the (R)-enantiomer and the racemic mixture of γ-decalactone as odor stimuli, numerous olfactory receptor neurons (ORNs) responding to both stimuli were found. No neurons responded preferentially to the racemic mixture, showing that these beetles seem to lack receptors specific for the (S)-enantiomer. The enantiomeric specificity of ORNs was confirmed by gas chromatography-linked single-sensillum recordings where the two enantiomers in a racemic mixture were separated on a chiral column. Furthermore, in field experiments that used the (R)-enantiomer and the racemic mixture as lures, the attraction of O. eremita females corresponded to the amount of (R)-enantiomer released from lures with the (S)-enantiomer displaying no antagonistic effects. Trap catch data also suggested that the (S)-enantiomer is not a behavioral antagonist for E. ferrugineus. The odor-based system can be highly efficient in attracting the larval predator where trap catch in 1 yr almost equaled the total number of specimens collected in Sweden until 1993. Our study shows that racemic γ-decalactone could be used for cost-effective monitoring of both beetles.     

Chiral esters: Sex pheromone of the bagworm,Oiketicus kirbyi (Lepidoptera: Psychidae)

Gas chromatographic-electroantennographic detection (GC-EAD) analyses of pheromone extract of female bagworms,Oiketicus kirbyi (Guilding), revealed five EAD-active compounds. Retention index calculations, GC-mass spectrometry in both full-scan and selected-ion monitoring modes and GC-EAD analyses of authentic standards identified the compounds as 1-methylbutyl octanoate (MBO), 1-methylbutyl nonanoate (MBN), 1-methylbutyl decanoate (MBD), 1-methylpentyl decanoate (MPD), and 1-methylbutyl dodecanoate (MBDD). Of these five chiral esters, MBD was most abundant in extracts and elicited the strongest antennal response. In field experiments in Costa Rica, (R)-MBD attractedO. kirbyi males, whereas (S)-MBD in combination with (R)-MBD inhibited response.R but notS enantiomers of MBO, MBN, and MBDD strongly synergized attraction to (R)-MBD. (S)-MBO and (S)-MBDD were inactive, whereas (S)-MBN was inhibitory. (R)-, (S)- and racemic MPD were inactive. Blends of (R)-MBD in ternary combination with either (R)-MBO and (R)-MBN or (R)-MBN and (R)-MBDD were as attractive as the five-ester blend. Five- and four-ester blends were equally attractive, suggesting redundancy of pheromone components for attraction of males. The multiple sex pheromone component blend of chiral esters inO. kirbyi may have evolved to maintain species-specific communication in bagworm communities of tropical Americas.     

Sex Pheromone Components of Indian Gypsy Moth, <Emphasis Type="Italic">Lymantria obfuscata</Emphasis>

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.     

Chemical Characterization of Territorial Marking Fluid of Male Bengal Tiger, <Emphasis Type="Italic">Panthera tigris</Emphasis>

The territorial marking fluid of the male Bengal tiger, Panthera tigris, consists of a mixture of urine and a small quantity of lipid material that may act as a controlled-release carrier for the volatile constituents of the fluid. Using gas chromatography and gas chromatography–mass spectrometry, 98 volatile compounds and elemental sulfur were identified in the marking fluid. Another 16 volatiles were tentatively identified. The majority of these compounds were alkanols, alkanals, 2-alkanones, branched and unbranched alkanoic acids, dimethyl esters of dicarboxylic acids, γ- and δ-lactones, and compounds containing nitrogen or sulfur. Several samples of the marking fluid contained pure (R)-3-methyl-2-octanone, (R)-3-methyl-2-nonanone, and (R)-3-methyl-2-decanone, but these ketones were partly or completely racemized in other samples. The γ-lactone (S)-(+)-(Z)-6-dodecen-4-olide and the C₈ to C₁₆ saturated (R)-γ-lactones and (S)-δ-lactones were present in high enantiomeric purities. The chiral carboxylic acids, 2-methylnonanoic acid, 2-methyldecanoic acid, 2-methylundecanoic acid, and 2-ethylhexanoic acid were racemates. Cadaverine, putrescine, and 2-acetylpyrroline, previously reported as constituents of tiger urine, were not detected. The dominant contribution of some ketones, fatty acids, and lactones to the composition of the headspace of the marking fluid suggests that these compounds may be important constituents of the pheromone. Although it constitutes only a small proportion, the lipid fraction of the fluid contained larger quantities of the volatile organic compounds than the aqueous fraction (urine). The lipid derives its role as controlled-release carrier of the chemical message left by the tiger, from its affinity for the volatiles of the marking fluid. Six proteins with masses ranging from 16 to 69 kDa, inter alia, the carboxylesterase-like urinary protein known as cauxin, previously identified in the urine of the domestic cat and other felid species, were identified in the urine fraction of the marking fluid. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Homofarnesals: Female Sex Attractant Pheromone Components of the Southern Cowpea Weevil, <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis>

The southern cowpea weevil, Callosobruchus chinensis (Coleoptera: Bruchidae), is a major pest of stored legumes in warm temperate and tropical climates. The female sex attractant pheromone was extracted from filter-paper shelters taken from containers that housed virgin females. The extracts were purified by various chromatographic techniques, and the biologically active components in the fractions were screened by gas chromatographic–electroantennographic detection analysis with male antennae. Two compounds that elicited electrophysiological responses were isolated, and gas chromatography–mass spectrometry, nuclear magnetic resonance, and micro-chemical analyses suggested that the active compounds were homofarnesals, (2Z,6E)- and (2E,6E)-7-ethyl-3,11-dimethyl-2,6,10-dodecatrienals. Males of C. chinensis were significantly attracted to filter paper discs loaded with the synthetic compounds at 0.01–0.1 ng compared to solvent control in a Y-tube olfactometer assay. These pheromone components represent unique chemical structures within the genus Callosobruchus.     

Aggregation pheromone of square-necked grain beetle,Cathartus quadricollis (Guér.)

When feeding on rolled oats, male square-necked grain beetles,Cathartus quadricollis (Guér.), produced the aggregation pheromone (3R,6E)-7-methyl-6-nonen-3-yl acetate, for which the trival name quadrilure is proposed. The pheromone was highly attractive to both sexes in a two-choice, pitfall olfactometer modified to retain responding beetles by placing a food stimulus (an oat flake) in the glass vials containing the experimental and control stimuli. TheS enantiomer of the pheromone was inactive. Males also produced small amounts of (E)-7-methyl-6-nonen-3-one, (E)-7-methyl-6-nonen-3-ol, and (6E)-7-methyl-3-propyl-2,6-nonadienyl acetate, but these compounds were inactive in the laboratory bioassay. Segregated males and females both produced (R)-(–)-1-octen-3-ol, which by itself was repellent to both sexes but did not diminish beetle response to the aggregation pheromone.Coleoptera: Cucujidae.Research supported by the Natural Sciences and Engineering Research Council of Canada, Strategic Grant G1039 and Operating Grants A3881 and A3706.