A new component of the female sex pheromone ofBlattella germanica (L.) (Dictyoptera: Blattellidae) and interaction with other pheromone components

A fourth component, 3,11-dimethyi-2-heptacosanone, was identified as a cuticular contact sex pheromone of the female German cockroach,Blattella germanica. In behavioral assays, higher dosages of 3,11-dimethyl-2-heptacosanone were needed to elicit similar sexual responses in males to those elicited by the major pheromone component, 3,11-dimethyl-2-nonacosanone. A 1585 blend of the C₂₇ and C₂₉ methyl ketone homologs resulted in a dose-response curve intermediate between that of each of the components alone, indicating independence of activity of each component and lack of synergism. Moreover, the activity of 3,11-dimethyl-2-nonacosanone was not enhanced by female cuticular hydrocarbons. The relationship between sexual responses of males to females and to isolated female antennae, and the amount of cuticular pheromone on whole females was investigated. Cuticular sex pheromone found on females increased with the age of the female, as did the male response to whole females. However, a bimodal male response was elicited by isolated female antennae. Differences between behavioral and analytical assays of pheromone are discussed.     

New Contact Sex Pheromone Components of the German Cockroach, Blattella germanica, Predicted from the Proposed Biosynthetic Pathway

Upon contacting the cuticle of a sexually mature female, a male German cockroach exhibits a characteristic courtship behavior: he turns away from the female and raises his wings, thereby exposing tergal glands. The glandular secretion stimulates the female to mount the male and feed, thus positioning her appropriately for copulation. A multi-component contact sex pheromone produced by females is responsible for eliciting courtship behavior. The most abundant pheromone components are 3,11-dimethylnonacosan-2-one and 3,11-dimethylheptacosan-2-one, oxidation products of the abundant hydrocarbon analogs 3,11-dimethylnonacosane and 3,11-dimethylheptacosane, respectively. The C₂₉-dimethyl ketone is thought to be further metabolized to two less abundant pheromone components, 29-hydroxy-3,11-dimethylnonacosan-2-one and 29-oxo-3,11-dimethylnonacosan-2-one. Based on this proposed biosynthetic pathway of pheromone production, we hypothesized that 3,11-dimethylheptacosan-2-one also would be oxidized to give two candidate pheromone components, 27-hydroxy-3,11-dimethylheptacosan-2-one, and 27-oxo-3,11-dimethylheptacosan-2-one. By using bioassay-guided fractionation and chemical analyses of cuticular extracts of virgin females and synthesis of the (3S,11S)-isomer of each of the two predicted pheromone components, we showed that the epicuticle of the German cockroach does indeed contain these two compounds. The contact sex pheromone of the female German cockroach, thus may consist of at least six biosynthetically related components.     

Cuticular hydrocarbons ofAedes hendersoni cockerell andA. triseriatus (SAY)

Field-caught adult male and femaleAedes hendersoni are difficult to distinguish from the sibling speciesA. triseriatus. We found that mosquitoes from the same sex of the sibling species can not be readily separated either by unique cuticular hydrocarbon components or by differences in percent composition of those components. Multivariate analysis of the cuticular hydrocarbon data does not provide good separation. Cuticular hydrocarbons were identified using gas chromatography electron-impact mass spectrometry and gas chromatography chemical-ionization mass spectrometry. Flame-ionization capillary gas chromatography was used for quantitative analysis of individual mosquitoes. Sixty-four hydrocarbons with chain lengths from C₁₆ to greater than C₄₆ were common to both species. Identified hydrocarbon components weren-alkanes, monomethylalkanes, dimethylalkanes, trimethylalkanes, and alkenes.Diptera: Culicidae.     

Courtship Pheromones in Parasitic Wasps: Comparison of Bioactive and Inactive Hydrocarbon Profiles by Multivariate Statistical Methods

Cuticular hydrocarbons play a significant role in the regulation of cuticular permeability and also in the chemical communication of insects. In the parasitoid Lariophagus distinguendus (Hymenoptera: Pteromalidae), male courtship behavior is mediated by a female-produced sex pheromone. Previous studies have shown that the chemicals involved are already present in the pupal stage of both males and females. However, pheromonal activity in males decreases shortly after emergence. This pheromonal deactivation occurs only in living males, suggesting an active process rather than simple evaporation of bioactive compounds. Here, we present evidence that the sex pheromone of L. distinguendus is composed of a series of cuticular hydrocarbons. Filter paper disks treated with nonpolar fractions of cuticular extracts of freshly emerged males and females, 72-hr-old females, and yellowish pupae caused arrestment and stimulated key elements of courtship behavior in males, whereas fractions of 72-hr-old males did not. Sixty-four hydrocarbons with chain length between C₂₅ and C₃₇ were identified in the fractions by gas chromatography-mass spectrometry (GC-MS). Methyl-branched alkanes with one to four methyl groups were major components, along with traces of n-alkanes and monoalkenes. Principal component analysis, based on the relative amounts of the compounds, revealed that cuticular hydrocarbon composition differed among all five groups. By using partial least squares-discriminant analysis, we determined a series of components that differentiate bioactive and bioinactive hydrocarbon profiles, and may be responsible for pheromonal activity of hydrocarbon fractions in L. distinguendus.     

Female sex pheromone of the german cockroach,Blattella germanica (L.) (Orthoptera: Blattellidae), responsible for male wing-raising II. 29-Hydroxy-3,11-dimethyl-2-nonacosanone

One component of the sex pheromone responsible for wing-raising in courtship behaviors ofBlattella germanica males was isolated from cuticular wax of the mature females and identified as 29-hydroxy-3,11-dimethyl-2-nonacosanone. This component showed the activity at a concentration as low as 3 g/ml in carbon tetrachloride on the behavioral bioassay.     

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.     

Contact Sex Pheromone Components of the Cowpea Weevil, Callosobruchus maculatus

The cowpea weevil, Callosobruchus maculatus, is a major pest of stored pulses. Females of this species produce a contact sex pheromone that elicits copulation behavior in males. Pheromone was extracted from filter-paper shelters taken from cages that housed females. Crude ether extract stimulated copulation in male C. maculatus. Initial fractionation showed behavioral activity in acidic and neutral fractions. Furthermore, bioassay-guided fractionation and gas chromatography-mass spectroscopy (GC-MS) analysis of active fractions revealed that the active components of the acidic fraction were 2,6-dimethyloctane-1,8-dioic acid and nonanedioic acid. These components along with the hydrocarbon fraction, a mixture of C₂₇–C₃₅ straight chain and methyl branched hydrocarbons, had a synergistic effect on the behavior of males. Glass dummies treated with an authentic pheromone blend induced copulation behavior in males. The potential roles of the contact sex pheromone of C. maculatus are discussed.     

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.     

Mating stimulant pheromone and cuticular lipid constituents ofFannia femoralis (Stein) (Diptera: Muscidae)

The cuticular lipids of male and femaleFannia femoralis were similar for recently emerged insects but soon began to develop chromatographic patterns characteristic of each sex. Mature females contained more C₃₁ and C₃₃ monoolefin in the cuticular lipid than males. Also, the double bonds in the monoolefins of the female lipid were situated predominantly at the eleventh and thirteenth carbons, while most of those from the males were centrally located in the molecule or at the ninth carbon.The female C₃₁ monoolefin stimulated copulation by the males, but more mating activity occurred when the saturated hydrocarbons present in the female cuticular lipids were added. The synthetic monoolefin most active as a mating stimulant pheromone was (Z)-11-hentriacontene, but the addition of female alkanes or of syntheticn-alkanes to (Z)-11-hentriacontene increased the activity of the synthetic pheromone.A portion of a dissertation intended for submission by the first author to the Graduate School of the University of Maryland in partial fulfillment of the requirements for the Ph.D. degree.Mention of a proprietary or commercial product in this paper does not constitute an endorsement of this product by the U.S. Department of Agriculture or the University of Maryland.     

Cuticular Hydrocarbons of Buffalo Fly, <Emphasis Type="Italic">Haematobia exigua</Emphasis>, and Chemotaxonomic Differentiation from Horn Fly, <Emphasis Type="Italic">H. irritans</Emphasis>

We determined the quantity and chemical composition of cuticular hydrocarbons of different strains, sexes, and ages of buffalo flies, Haematobia exigua. The quantity of cuticular hydrocarbons increased from less than 1 μg/fly for newly emerged flies to over 11 μg/fly in 13-d-old flies. The hydrocarbon chain length varied from C₂₁ to C₂₉, with unbranched alkanes and monounsaturated alkenes the major components. Newly emerged flies contained almost exclusively C₂₇ hydrocarbons. Increasing age was accompanied by the appearance of hydrocarbons with shorter carbon chains and an increase in the proportion of alkenes. 11-Tricosene and 7-tricosene were the most abundant hydrocarbons in mature H. exigua. Cuticular hydrocarbons of H. exigua are distinctly different from those of horn flies, Haematobia irritans. The most noticeable differences were in the C₂₃ alkenes, with the major isomers 11- and 7-tricosene in H. exigua and (Z)-9- and (Z)-5-tricosene in H. irritans, respectively. Cuticular hydrocarbon analysis provides a reliable method to differentiate the two species, which are morphologically difficult to separate. The differences in cuticular hydrocarbons also support their recognition as separate species, H. exigua and H. irritans, rather than as subspecies.     

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.     

Cuticular hydrocarbons of the eastern subterranean termite,Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae)

Major cuticular hydrocarbon components in several castes ofReticulitermes flavipes (Kollar) have been identified and quantitated. Types of hydrocarbons present includen-alkanes, 2-methylalkanes, 3-methylalkanes, 5-methylalkanes, an alkene, and an alkadiene, with a range in carbon numbers from C₂₁ to C₂₆, This is the first report on insect cuticular hydrocarbons in which both 2- and 3-methylalkanes are present, as well as the first report of an insect with a conjugated alkadiene.     

Role of cuticular hydrocarbons of aphid parasitoids in their relationship to aphid-attending ants

Lysiphlebus cardui, the dominant aphidiid parasitoid of the black bean aphid,Aphis fabae cirsiiacanthoidis (Afc), on creeping thistle, is able to forage in ant-attended aphid colonies without being attacked by ants. Several behavioral observations and experimental studies led to the hypothesis thatL. cardui mimics the cuticular hydrocarbon profile of its host aphid. Chemical analysis of the cuticular extracts revealed that bothL. cardui and Afc exclusively possess saturated hydrocarbons:n-alkanes, monomethyl (MMA), dimethyl (DMA), and trimethyl alkanes (TMA). Comparison of the hydrocarbon profiles of parasitoid and aphid showed great qualitative resemblance between parasitoid and host:L. cardui possesses almost all host-specific compounds in addition to species-specific hydrocarbons of mainly higher molecular weight (>C₃₀). However, there is a lesser quantitative correspondence between parasitoid and host aphid. Furthermore, we analyzed the cuticular hydrocarbon profile of another parasitoid of Afc,Trioxys angelicae. This aphidiid species is vigorously attacked and finally killed by honeydewcollecting ants when encountered in aphid colonies. Its cuticular hydrocarbon profile is characterized by the presence of large amounts of (Z)-11-alkenes of chain lenghts C₂₇, C₂₉, C₃₁, and C₃₃, in addition to alkanes and presumably trienes. The role of the unsaturated hydrocarbons onT. angelicae as recognition cues for aphid-attending ants is discussed.     

Cuticular hydrocarbons regulate mate recognition,male aggression,and female choice of the rove beetle,Aleochara curtula

Immature, starved, or multiply mated males of the staphylinid beetle,Aleochara curtula, mimic their females chemically. The titer of the female sex pheromone components (Z)-7-heneicosene and (Z)-7-tricosene was quantified for various physiological types and both sexes by gas chromatography and correlated with the sexual response of males towards the cuticular hydrocarbon fractions. Modulation of intermale aggression by production of the female pheromone was shown by (1) reduction of the alkene titer of females kept at elevated temperatures, (2) treating live males with the synthetic female pheromone mixture, and (3) gradual amputation of male antennal segments.A. curtula males do not fight against members of otherAleochara species with a different hydrocarbon pattern. Contamination ofA. peschkei males with the hydrocarbon fraction ofA. curtula males, however, provoked the release of aggression. Choosy females reject mating attempts of males bearing the female sex pheromone.     

Syntheses of female sex pheromone precursors of pine sawfly species and of some structurally related methyl-branched long-chain 2-alkanols

3,7-Dimethyl-2-undecanol, 3,7,9-trimethyl-2-tridecanol, and 3,7, 11-trimethyl-2-tridecanol were synthesized as racemic mixtures in moderate yields. The alcohols are known precursors of the female sex pheromones of the pine sawfly species Diprion nipponica, Macrodiprion nemoralis, and Microdiprion pallipes, respectively. Stereoisomeric mixtures of 3,8,12-trimethyl-2-tridecanol, erythro-(2R,3R,11R/S)-3,11-dimethyl-2-tetradecanol, 3,5-dimethyl-2-tetradecanol, and 5,7-dimethyl-2-tetradecanol, structurally related to sex pheromone alcohol precursors of pine sawfly species, were also synthesized in moderate yields. The key reaction in the syntheses was the ring opening of -butyrolactones by using different alkyl lithiums as nucleophiles.     

Cuticular hydrocarbons ofReticulitermes virginicus (Banks) and their role as potential species- and caste-recognition cues

The cuticular hydrocarbon components of four castes ofReticulitermes virginicus (Banks) have been identified and quantitated. Components identified includen-alkanes; 2-, 3-, 11-, 13-, and 15-methyl-alkanes; 11,15-dimethylalkanes, (Z)-9-alkenes; (Z,Z)-7,9-dienes; and (E/Z)-6,9-dienes ranging in carbon number from C₂₁ to C₄₀. All caste forms ofR.virginicus contained the same components, but showed caste-specific proportions. Comparison of these hydrocarbons with those of the sympatric termiteR. flavipes (Kollar) suggest that cuticular hydrocarbons might serve as species- and caste-recognition cues. A bioassay was developed to test this species-recognition hypothesis, with the experimental results supporting the hypothesis.Isoptera: Rhinotermitidae.     

Characterization of a Female-Produced Courtship Pheromone in the Parasitoid Nasonia vitripennis

Males of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) show a characteristic courtship behavior. We demonstrate that male arrestment and key behavioral elements of the courtship sequence are mediated by a female-derived contact sex pheromone. Males were arrested on paper disks treated with female extracts but not on those treated with male extracts. Male responsiveness was influenced by the surface to which female extracts were applied. Extracts applied to an extracted beetle elytron arrested males more strongly than those applied to filter paper of comparable size. However, more complex behavioral elements, such as head nodding and copulation attempts, were shown only when extracts were applied to extracted male cadavers, suggesting that tactile or visual cues synergize the male response. The chemicals involved are stable, of low volatility, and nonpolar. Dead females arrested males and elicited courtship behavior for at least 8 d. Males showed no sign of attraction to live females at a distance of 3 cm in an olfactometer. Fractionation of female extracts demonstrated that the activity was exclusively located in the nonpolar fraction. Analysis of the active fraction by gas chromatography–mass spectrometry revealed that cuticular hydrocarbons with chain lengths between 25 and 37 carbon units were present. Comparison of hydrocarbon profiles from males and females showed qualitative and quantitative differences. These results suggest that sex-specific cuticular hydrocarbons are the key signals mediating the male courtship behavior in N. vitripennis.     

(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.Electronic supplementary material Supplementary material to this paper is available in electronic form at.     

Hydrocarbons ofNasutitermes acajutlae and comparison of methodologies for sampling cuticular hydrocarbons of caribbean termites for taxonomic and ecological studies

Using data from the arboreal nestingNasutitermes acajutlae (Holmgren), we propose standard collection and extraction methodology for characterization of cuticular hydrocarbons of termites under field conditions in the tropics. Specifically, we evaluated: (1) the effect of the duration and the number of extractions; (2) the effect of drying termites before extraction; (3) the effect of sample size; (4) the effect of solvents (ethanol versus hexane) on cuticular hydrocarbon profiles. Olefins comprise ca. 70% of the cuticular hydrocarbons ofN. acajutlae. Hydrocarbons consist of two distinct groups: early-eluting components, primarilyn-alkanes and methyl-branched alkanes, and late-eluting compounds, which consist almost exclusively of unsaturated components with one to six double bonds. Soldiers have more early-eluting compounds than workers or alates. Nests from the same island had qualitatively similar, but quantitatively dissimilar hydrocarbon mixtures. Brief extractions of 300 live workers in 10 ml of hexane for only 20 sec produced a hydrocarbon mixture equivalent to a 10-min extraction. Long-term extraction of 300 workers in hexane for two years resulted in different mixtures of hydrocarbons. Drying workers tended to enhance extraction of the less abundant unsaturated compounds such as C_41.4 and C_41.5. A single extraction of a minimum of 100 workers (live or dried), with hexane for 20 sec to 10 min is best; these extraction regimes resulted in mixtures of hydrocarbons that are quantitatively very similar. For quantitative comparisons, extracts from dried samples should not be compared to those from live samples. Storage in ethanol caused numerous unidentified, nonhydrocarbon compounds to be extracted either from the cuticle or from internal tissues.     

The Egg Parasitoid <Emphasis Type="Italic">Trissolcus basalis</Emphasis> uses <Emphasis Type="Italic">n</Emphasis>-nonadecane,a Cuticular Hydrocarbon from its Stink Bug Host <Emphasis Type="Italic">Nezara viridula</Emphasis>, to Discriminate Between Female and Male Hosts

Contact kairomones from adult southern green stink bugs, Nezara viridula (L.) (Heteroptera: Pentatomidae) that elicit foraging behavior of the egg parasitoid Trissolcus basalis (Wollaston) were investigated in laboratory experiments. Chemical residues from tarsi and scutella of N. viridula induced foraging by gravid female T. basalis. Residues from body parts of female N. viridula elicited stronger responses than those from the corresponding body parts of males. Deproteinized tarsi still elicited searching responses from wasps, indicating that the kairomone was not proteinaceous. Hexane extracts of host cuticular lipids induced searching responses from T. basalis, with a strong preference for extracts from female hosts. Extracts consisted primarily of linear alkanes from nC₁₉ to nC₃₄, with quantitative and qualitative differences between the sexes. Extracts of female N. viridula contained more nC₂₃, nC₂₄, and nC₂₅ than the corresponding extracts from males, whereas nC₁₉ was detected only in extracts from males. Direct-contact solid phase microextraction (DC-SPME) of N. viridula cuticle and of residues left by adult bugs walking on a glass plate confirmed gender-specific differences in nC₁₉. Trissolcus basalis females responded weakly to a reconstructed blend of the straight-chain hydrocarbons, suggesting that minor components other than linear alkanes must be part of the kairomone. Addition of nC₁₉ to hexane extracts of female N. viridula significantly reduced the wasps’ arrestment responses, similar to wasps’ responses to hexane extracts of male hosts. Overall, our results suggest that a contact kairomone that elicits foraging by T. basalis females is present in the cuticular lipids of N. viridula, and that the presence or absence of nC₁₉ allows T. basalis females to distinguish between residues left by male or female hosts. The ecological significance of these results in the host location behavior of scelionid egg parasitoids is discussed.