Formation of Colony Odor in Ponerine Ant Pachycondyla apicalis

The mechanisms operating in the formation and maintenance of colony odor in the ponerine ant Pachycondyla apicalis were studied using radioactive tracers. Using [l-¹⁴C]acetate as a precursor, the de novo biosynthesis and distribution of pentane-extractable lipids within the ant's body were followed. Twenty-four hours after injection, newly synthesized alkanes, alkenes, as well as more polar lipids were found in the postpharyngeal gland, the epicuticle, and internally. Ants that had their mouths blocked with beeswax possessed significantly less radiolabeled lipids (all classes measured) in the postpharyngeal gland but had augmented levels in the internal pool and the epicuticle. Both hydrocarbons and more polar lipids were exchanged between the respective postpharyngeal glands and the epicuticle. The transfer to postpharyngeal glands was highest when both ants had an open mouth or when the recipient ant had an open mouth but the donor's mouth was blocked. This suggests that the transfer to the postpharyngeal gland in this species is by allogrooming and not by trophallaxis. Transfer to the cuticle was low and comparable in all treatments. Behavioral observations during the first 6 hr of the dyadic encounters and in intact colonies confirmed that the ants did not engage in trophallaxis. The level of transfer to the postpharyngeal gland in P. apicalis was significantly lower than in representatives of other Formicidae subfamilies studied so far. We attribute this difference to the evolution of trophallaxis in the higher Formicidae.     

Identification of a novel moth sex pheromone inEriocrania cicatricella (Zett.) (Lepidoptera: Eriocraniidae) and its phylogenetic implications

Extracts from different body parts of adult femaleEriocrania cicatricella (Zett.) were tested for electrophysiological activity on conspecific male antennae. Extracts from the Vth abdominal segment, containing a pair of exocrine glands, elicited the largest electroantennographic response when compared to extracts of other body parts. Female extracts were analyzed by gas chromatography with simultaneous flame ionization and electroantennographic detection (EAD). The EAD active peaks were identified as (Z)-4-hepten-2-one, (2R)-heptane-2-ol, and (2R)-(Z)-4-hepten-2-ol by coinjection on a gas chromatography and by comparison of mass spectra with those of synthetic standards. In field tests, a blend of these three pheromone components was highly attractive to conspecific males, and a subtractive assay confirmed that the unsaturated alcohol is the major pheromone component, whereas no definite behavioral activity could be assigned to the ketone or the saturated alcohol. A bait containing the two alcohols withS-configuration was attractive to maleE. sparrmannella (Bosc), whereas no males ofE. cicatricella were found in these traps. The sex pheromone compounds inE. cicatricella are chemically similar to pheromones reported in Trichoptera and they are produced in homologous glands.     

Aldehydic contact poisons and alarm pheromone of the antCrematogaster scutellaris (Hymenoptera: Myrmicinae)

The Dufour gland ofCrematogaster scutellaris stores a mixture of long-chain primary acetates bearing a cross-conjugated dienone (Scheme 1, la-c). The poison gland contains two highly active enzymes: an acetate esterase and an alcohol oxidase. During venom emission, the constituents of both glands mix and accumulate on the sting, where the formation of the highly electrophilic aldehydes (Scheme 1, 2a-c) from their acetate precursors is initiated. Acetic acid, produced during the reaction, acts as alarm pheromone. The toxicity of the acetates (Scheme 1, la-c) and of the crude secretion has been assessed by topical application onMyrmica rubra. The acetatecontaining secretion from the Dufour gland was less toxic than the enzymatically altered secretion that was rich in aldehydes. The production of acids (Scheme 1, 3a-c) was an artifact resulting from the nonenzymatic oxidation of the unstable aldehydes.     

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.     

Production and release of (Z,E)-9,12-tetradecadienal by sex pheromone glands of females ofPlodia interpunctella (lepidoptera: pyralidae)

Extracts of sex pheromone glands obtained from females ofPloida interpunctella contained detectable amounts of (Z,E,)-9,12-tetradecadien-1-ol acetate (Z9,E12–14:Ac) and (Z,E.)-9,12-tetradecadien-1-ol (Z9,E12–14:OH) 4 hr prior to the first scotophase after adult emergence. The amount of pheromone increased during the first 4 hr of the scotophase and then declined to low levels during the subsequent photophase. Decapitation of females immediately after emergence, prior to expansion of the wings, inhibited production of pheromone during the subsequent 48 hr. Injection of extracts of the heads of 1-day-old females ofP. interpunctella of partially purified extracts of the cephalic ganglia of females of the corn earworm moth into decapitated females stimulated production of bothZ9,E12–14:Ac andZ9,E12–14:OH as well as production of (Z,E)-9,12-tetradecadienal (Z9,E12–14:Al). This aldehyde was subsequently identified from extracts of pheromone glands obtained from naturally calling females as well as from volatiles emitted by calling females. Studies on the terminal steps in biosynthesis of the pheromone showed thatZ9,E12–14:OH was produced from the corresponding acetate and thatZ9,E12–14:Al was produced from the alcohol via the action of an oxidase(s).     

Volatile compounds from the predatory insectPodisus maculiventris (Hemiptera: Pentatomidae)

In the predatory spined soldier bug,Podisus maculiventris, the dorsal abdominal glands are much smaller in adult females than males. Females produce a mixture of (E)-2-hexenal, (E)-2-octenal, (E)-2-hexenoic acid, benzaldehyde, and nonanal in these glands. The female dorsal abdominal gland secretion may be a close-range pheromone since the dorsal abdominal gland secretion from males has been shown to be a long-range aggregation pheromone in this species. The metathoracic scent gland secretions of male and female spined soldier bugs are apparently identical, and similar to that of other pentatomids, except for the presence of the monoterpene alcohol, linalool.Mention of a commercial product does not constitute an endorsement of this product by the USDA.     

The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones

Many moth pheromones are composed of mixtures of acetates of long‐chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl‐CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non‐insect acetyltransferase alternative, we expressed a plant‐derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with EaDAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27‐ and 10‐fold higher in vivo and in vitro efficiency, respectively, compared to EaDAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.     

Salivary glands and preputial glands of males as source of estrus-stimulating pheromone in female mice

Male mice produce pheromones that induce estrus in adult female mice. The synthesis of these chemosignals is controlled by testosterone. Male tissues abundant in testosterone-binding receptors were tested for their ability to stimulate estrus in grouped females. The percentage of females that showed estrus during two days following exposure to male tissues was used as the indicator of biological activity of tested samples. Homogenates of salivary glands and preputial glands induced estrus; other tested organs (liver, testes, coagulation and vesicular glands or kidneys) did not stimulate estrus in female mice. These results indicate that estrus-stimulating pheromone has multiple sources.     

Reevaluation of the Role of Mandibular Glands in Regulation of Reproduction in Bumblebee Colonies

Possible pheromonal control of worker reproduction was tested in Bombus terrestris. The mode of assay included exposure of callow workers to extracts originating from different queen parts and measuring the effect on the in vitro biosynthesis of juvenile hormone (JH), the apparent gonadotropin in this species. Both queen total body extracts applied to dummies consisting of oven-dried or Soxhlet-washed virgin queen bodies and cuticular washes applied to living virgin queens effectively inhibited the biosynthesis of JH in callow workers. None of the five exocrine glands (mandibular, hypopharyngeal, salivary, Dufour's, and tarsal) demonstrated inhibitory activity. Likewise, the use of synthetic 3-hydroxy acids, found in queen mandibular glands, were ineffective in blocking JH biosynthesis in queenless workers. The results suggest that the queen may use a primer pheromone spread on the epicuticle as a means to inhibit worker reproduction. However, our results are not consistent with the prevailing hypothesis that in B. terrestris the main source of the pheromone that inhibits worker reproduction is in the queen's mandibular glands.     

Kairomone from dandelion,Taraxacum officinale,attractant for scarab beetleAnomala octiescostata

The attraction of the scarab beetleAnomala octiescostata to dandelion,Taraxacum officinale, was demonstrated to be chemically mediated by a mixture ofcis-3-hexenyl acetate, benzaldehyde, phenylacetaldehyde, benzyl alcohol, phenethyl alcohol, phenylacetonitrile, and benzyl benzoate, in the ratio 4:8:14:3:5:19:11. Combination of the synthetic kairomone and sex pheromone (buibuilactone + japonilure, 8:2), significantly increased the total catches ofA. octiescostata. Catches of male (but not female) beetles were significantly higher with the kairomone-pheromone blend than with kairomone alone. The synergistic effect of the kairomone from dandelion on the attractiveness did not significantly differ from that of a food-type lure, anethol, geraniol, and phenethyl propionate (9:0.5:0.5). The latter combined with the synthetic sex pheromone resulted in better attraction of female (but not male)A. octiescostata than the sex pheromone alone.     

(3Z,6Z,8E)-3,6,8-Dodecatrien-1-ol: Sex pheromone in a higher fungus-growing termite,Pseudacanthotermes spiniger (Isoptera,macrotermitinae)

The female sex pheromone of the fungus-growing termitePseudacanthotermes spiniger (Termitidae, Macrotermitinae) was isolated from sternal glands of alates. The compound inducing attraction and excitation in males was identified as the (3Z,6Z,8E)-3,6,8-dodecatrien-1-ol by GC-MS, microhydrogenation, GC-FTIR, and NMR. This unsaturated alcohol is present in both sexes but in much higher quantities in females than in males (about 10 times). The hypothesis is suggested that this alcohol, which is detected at extremely low concentrations by the workers ofP. spiniger, may be used either as a trail-following pheromone or a sex pheromone according to concentrations and to target castes. The presence of this alcohol in Macrotermitinae reinforces the idea of a phylogenetic proximity between this subfamily of higher termites and the lower termites Rhinotermitidae, where the unsaturated alcohol was previously found. The sternal glands of alates ofP. spiniger also contain a geometric isomer of (3Z,6Z,8E)-3,6,8-dodecatrien-1-ol.     

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.     

Synthesis and pheromonal activity of 6,10,13-trimethyl-1-tetradecanol for predatory stink bug,Stiretrus anchorago (Heteroptera: Pentatomidae)

Racemic 6,10,13-trimethyl-1-tetradecanol has been synthesized. Successive elaboration of the molecule using 5-methyl-2-hexanone as the carbon-terminal fragment gave the desired alcohol in a 4% overall yield over 13 steps. Neglecting stereochemistry, the product was shown to be identical to the alcohol previously identified from the sternal glands of maleStiretrus anchorago. It attracts both adult sexes and larvae of this insect in the field, confirming its postulated identity as an aggregation pheromone. The corresponding aldehyde is more than 10 times more concentrated in airbornetrapped samples (5–7% by gas chromatography) than in extracts of glandular setae (0.4–0.5%). This alcohol or its isovalerate ester has been found in other predaceous pentatomids, providing further evidence for pheromones in these species.     

Sex Pheromone of the Common Sheep Moth, Hemileuca eglanterina, from the San Gabriel Mountains of California

The sex pheromone of Hemileuca eglanterina from the San Gabriel Mountains, California, was determined to be a combination of E10,Z12-hexadeca-10,12-dien-1-yl acetate, E10,Z12-hexadeca-10,12-dien-1-ol, and E10,Z12-hexadeca-10,12-dienal. Ratios of the compounds in extracts of female pheromone glands varied around a mean of 100 : 48 : 1.1 of the acetate, alcohol, and aldehyde, respectively. Field trials with synthetic compounds indicated that the optimum ratio of alcohol to aldehyde was 10 : 1 and that this ratio was more critical than the ratio of either compound to the acetate. A synthetic blend of 100 : 10 : 1 acetatendash;alcoholndash;aldehyde was effective at attracting male moths in the field. Additional compounds found in both extract and aeration samples failed to significantly increase trap catches of male moths, although some of these minor components elicited responses from male moth antennae in coupled gas chromatography–electroantennography studies.     

(Z)-10-Heptadecen-2-one and other 2-ketones in the aggregation pheromone blend ofDrosophila martensis,D. buzzatii,andD. serido

(Z)-10-Heptadecen-2-one was identified as the major aggregation pheromone component inDrosophila martensis, D. buzzatii, andD. serido. Aliphatic 2-ketones were not detected in newly eclosed males or in virgin females of any age. The amount of (Z)-10-heptadecen-2-one in the ejaculatory bulb and on the cuticle of males increased with age. Only trace quantities of (Z)-10-heptadecen-2-one were transferred to the female during mating. 2-Tridecanone was found in the ejaculatory bulb and on the cuticle of maleD. martensis andD. buzzatii, but notD. serido. The amount of 2-tridecanone increased with age. Unexpectedly,2-tridecanone inhibited the aggregation activity of (Z)-10-heptadecen-2-one inD. buzzatii. InD. martensis, 2-tridecanone was significantly less attractive than controls but did not decrease the attractiveness of one fly equivalent of (Z)-10-heptadecen-2-one.D. serido males did not have any 2-tridecanone and the activity of (Z)-10-heptadecen-2-one was not diminished inD. serido by the presence of 2-tridecanone.     

Differences in oxidase and esterase activities involved in pheromone biosynthesis in two species ofChoristoneura

The released pheromone and the glandular lipids, labeled with [¹⁴C]acetate, were analyzed fromChoristoneura orae andChoristoneura fumiferana budworm moths by thin-layer chromatography and autoradiography. Radiolabeled lipids in the gland appeared to be identical in the two moths with both insects containing high amounts of 11-tetradecenyl acetates. In contrast, theC. orae moths released primarily labeled acetate ester and alcohol, and the spruce budworm moths (C. fumiferana) labeled aldehyde consistent with the known composition of their respective pheromones. The levels of the enzymes responsible for converting the acetate ester into aldehyde were found to be significantly lower in gland extracts fromC. orae moths than fromC. fumiferana moths. These results implicate an acetate esterase and an alcohol oxidase in controlling the composition of the pheromone blend released from closely related budworm species.     

Aggregation pheromones inDrosophila borealis andDrosophila littoralis

Mature males ofDrosophila borealis andD. littoralis (Diptera: Drosophilidae) produce pheromones that attract both males and females in a wind-tunnel bioassay. Ethyl tiglate is a major pheromone component in both species. Isopropyl tiglate is a minor component, as active as ethyl tiglate on an equal-weight basis, but less abundant in the flies. Both species respond to (Z)-9-heneicosene, a compound they do not possess, but which is a pheromone component in closely related species.D. borealis andD. littoralis are also able to discriminate among various heneicosene isomers. These responses to hydrocarbons may represent artifacts of evolution in this group. For both species, the pheromone was strongly synergized by an extract of fermenting aspen bark, a host material ofD. borealis. Benzyl alcohol was identified as one active component, although it did not account for all the activity of the extract. In nature, the flies probably respond to mixtures of fly-derived and host-derived volatiles.     

Analysis of Sex Pheromone Gland Content of Individual Symmetrischema tangolias by Means of Direct Gland Introduction into a Two-dimensional Gas Chromatograph

The amounts and ratios of the four constituents of the sex pheromone gland of the moth Symmetrischema tangolias were measured during a 24-hr dark–light cycle. A new approach was followed that involved the direct introduction of sex pheromone glands into the liner of a two-dimensional gas chromatograph (2D-GC) by using a special temperature programmable GC injector. With this method it was possible to examine individual glands without prior processing. This provides a clear advantage over other methods. The detection limit was 0.5 ng. The total amount of sex pheromone in the glands varied strongly from individual to individual (3.8–350 ng/female). The total sex pheromone amounts were significantly lower in the scotophase than in the photophase (62.2 and 101.5 ng/female, respectively). The ratios of the various gland constituents showed a symmetrical distribution, which did not fluctuate during the 24-hr dark–light cycle.     

Identification of new sex pheromone components inTrichoplusia ni,predicted from biosynthetic precursors

In addition to the previously identified components (Z)-7-dodecenyl acetate and dodecyl acetate, sex pheromone glands ofTrichoplusia ni release (Z)-5-dodecenyl acetate, 11-dodecenyl acetate, (Z)-7-tetradecenyl acetate, and (Z)-9-tetradecenyl acetate. Bioassays in a flight tunnel showed that a synthetic blend of these six compounds elicited complete flights to the source from 95% of the males tested and elicited hairpenciling responses at the end of the flights from 88% of the males tested. This blend was not significantly different from intact pheromone glands, which elicited complete flights to the source from 98% of the males tested and hairpenciling responses from 91% of the males tested. In contrast, the previously identified two-component blend elicited significantly fewer complete flights to the source (33%) and did not elicit hairpenciling responses from any of the males tested. The search for additional sex pheromone components was prompted by our previous identification of unusual fatty acyl moieties in the gland that seemed to be possible biosynthetic intermediates.     

Trail-Following Pheromones in Basal Termites, with Special Reference to Mastotermes darwiniensis

In the framework of an evolutionary study, trail pheromones have been studied in the most basal extant termite, Mastotermes darwiniensis (Mastotermitidae), and two other basal termites, the Termopsidae Porotermes adamsoni (Porotermitinae) and Stolotermes victoriensis (Stolotermitinae). Although workers of M. darwiniensis do not walk in single file while exploring a new environment under experimental conditions and are unable to follow artificial trails in ‘open field’ experiments, they do secrete a trail-following pheromone from their sternal glands. This unique behavior might reflect a primitive function of communication of the sternal gland. The major component of the pheromone appears to be the same in the three basal species: the norsesquiterpene alcohol (E)-2,6,10-trimethyl-5,9-undecadien-1-ol. This represents a new chemical category of trail-following pheromones for termites. The quantity of pheromone was estimated as 20 pg/individual in M. darwiniensis, 700 pg/individual in P. adamsoni, and 4 pg/individual in S. victoriensis. The activity threshold was 1 ng/cm in M. darwiniensis and 10 pg/cm in P. adamsoni. In M. darwiniensis, the trail pheromone was secreted by sternal gland 4 and to a lesser degree by sternal gland 3, sternal gland 5 being almost inactive. This study highlighted phylogenetic relationships between the Mastotermitidae and two subfamilies of the Termopsidae, the Porotermitinae and the Stolotermitinae. Furthermore, it indicated a heterogeneity within the Termopsidae, with Porotermitinae and Stolotermitinae on one hand, and Termopsinae on the other. Finally, Mastotermitidae and Termopsidae, with C14 trail pheromones, are clearly separated from the Kalotermitidae, Rhinotermitidae, and Termitidae that secrete C12 or C20 trail pheromones.