Non-alkaloidal constituents of Virola elongata bark

Eleven of the major non-polar constituents of the dried bark of Virola elongata were isolated. A new neolignan, virolongin, two new lignans, dihydrosesartemin and β-dihydroyangambin, as well as the neolignan, eusiderin, the lignans, epi-sesartemin, epi-yangambin and yangambin, the cis and trans isomers of 3,5,4′-trimethoxystilbene and sitosterol were identified. The structures of virolongin, dihydrosesartemin and β-dihydroyangambin were determined.     

Anti-leishmanial activity of neolignans from Virola species and synthetic analogues

Surinamensin, a neolignan isolated from Virola surinamensis, 3,4,5-trimethoxy-8-[2',6'-dimethoxy-4'-(E)-propenylphenoxy]-phenylpropane, a neolignan isolated from Virola pavonis, and 25 of its synthetic analogues or correlated substances with ether linkages and their corresponding C-8 sulphur and nitrogen analogues, were tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Some were active against L. donovani promastigotes at 30 microM but inactive against intracellular amastigotes. The natural neolignan from V. pavonis was active against promastigotes at 100 microM. The highest selective activity was found in those compounds with sulphur bridges. The beta-ketosulfide (3,4-dimethoxy)-8-(4'-methylthiophenoxy)-propiophenone produced 42% inhibition of L. donovani amastigotes in the liver of BALB/c mice at 100 mg/kg given once daily for five consecutive days (P>0.05).     

Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp. AS2C isolated from the consortium

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)-1,3-dichloropropene) was enriched from an enhanced soil. This mixedculture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presenceof a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)-1,3-D, respectively. (E)-1,3-D was degraded faster than (Z)-1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)-1,3-D to (Z)-1,3-D orthe (Z) form to the (E) form did not occur.     

Effect of (E4, Z6)-4,6-Hexadecadienal in attraction of Stathmopoda masinissa with its pheromone components of Korean population

Timely insecticidal application for Stathmopoda masinissa Meyrick (Lepidoptera: Stathmopodidae), is important, for reducing damage to persimmon (Diospyros kaki Thunb.), an important tree fruit cultivated in Korea. In this regard, the early and precise detection of adult S. masinissa is desirable. In this study, we report the effect of (E4,Z6)-4,6-hexadecadienal (E4,Z6-16Ald) with sex pheromone components in attracting S. masinissa males. The sex pheromone of S. masinissa in the Korean population comprised two components, (E4,Z6)-4,6-hexadecadienyl acetate (E4,Z6-16Ac) and (E4,Z6)-4,6-hexadecadienol (E4,Z6-16OH). It was shown that the E4,Z6-16Ald acts as a synergist of E4,Z6-16Ac for attracting S. masinissa in the Japanese population. To test whether E4,Z6-16Ald could be used as an attractant in the Korean population, the E4,Z6-16Ald with the two pheromone components was evaluated in attracting S. masinissa males. Electroantennography (EAG) assays were performed to determine the antennal responses of S. masinissa males to the two pheromone components and E4,Z6-16Ald tested. A field attraction test with a combination of pheromones and E4,Z6-16Ald was carried out for 3 years in three different regions in Korea. E4,Z6-16Ald elicited as high a response as the two pheromone components. A mixture of the two pheromone components and E4,Z6-16Ald and a mixture of E4,Z6-16Ac and E4,Z6-16Ald attracted more S. masinissa males than a mixture of E4,Z6-16Ac and E4,Z6-16OH, the pheromone of Korean population. This new pheromone lure formulation with E4,Z6-16Ald is expected to contribute to the precise detection of S. masinissa by luring males to pheromone-baited traps.     

Identification of a four-component sex pheromone blend in Bonagota cranaodes (Lepidoptera: Tortricidae)

Analysis of sex pheromone glands of the apple leafroller Bonagota cranaodes Meyrick by gas chromatography coupled with mass spectrometry or electroantennographic detection showed the presence of 14 structurally related acetates and alcohols of the chain length 10-18, including the main pheromone component (E,Z)-3,5-dodecadienyl acetate (E3,Z5-12Ac). Male antennae responded to the main compound, its Z,Z isomer, (E,Z) -3,5-tetradecadienyl acetate (E3,Z5-14Ac), and the monoenes (Z)-5-dodecenyl acetate (Z5-12Ac) and (Z)-9-hexadecenyl acetate (Z9-16Ac). Traps baited with a four-component blend of E3,Z5-12Ac, Z5-12Ac, E3,Z5-14Ac, and Z9-16Ac in a 100:5:5:100 ratio were significantly more attractive than the main compound alone. This improved trap lure is more suitable for monitoring population densities of B. cranaodes, and for detection of the onset of the seasonal flight period. A more complete pheromone blend is of importance also with respect to current attempts to develop mating disruption for control of this major pest of apple in Brazil.     

Sex pheromone analysis and effective attraction of males of the cabbage webworm,Hellula undalis,inhabiting the Mekong Delta of Vietnam

Hellula undalis is a harmful insect pest of green mustard in the Mekong Delta of Vietnam. In order to establish a tool for a sustainable pest control program, the sex pheromone of H. undalis inhabiting the Mekong Delta was examined. GC-EAD and GC–MS analyses of pheromone gland extracts from the virgin females elucidated three new components, (Z)-11-tetradecenyl acetate (Z11-14:OAc), (Z)-11-hexadecenal (Z11-16:Ald), and (11E,13E)-11,13-hexadecadien-1-ol, in addition to the known pheromone component (11E,13E)-11,13-hexadecadienal (E11,E13-16:Ald). Double bond positions of the two monoenyl components were determined by GC–MS analysis of the pheromone extract treated with dimethyl disulfide. On the other hand, GC–MS analysis of the female body extract detected the unsaturated hydrocarbon (3Z,6Z,9Z)-3,6,9-tricosatriene (Z3,Z6,Z9-23:H). Field examinations of their synthetic compounds indicated the significant role of E11,E13-16:Ald as a major component and a clear synergistic effect of the two monoenyl compounds as a minor component. Although the 3:3:7 mixture of Z11-14:OAc, E11-16:Ald, and E11,E13-16:Ald captured the largest number of males among the tested mixtures, the activity was still quite a bit lower than that of virgin females. However, the 3:3:7:1 mixture, which was prepared by adding a small amount of Z3,Z6,Z9-23:H to the 3:3:7 ternary lure, succeeded in attracting males more powerfully than the females did. This strong synergistic effect was not observed when the triene was added to unmixed E11,E13-16:Ald, indicating important roles of not only the triene but also the two monoenyl compounds as natural pheromone components.     

10,20-Methanorhodopsins: (7E,9E,13E)-10,20-methanorhodopsin and (7E,9Z,13Z)-10,20-methanorhodopsin. 11-cis-locked rhodopsin analog pigments with unusual thermal and photo-stability

Synthesis of the retinal analog, 10,20-methanoretinal (R6), where the 11Z conformation is locked in a six-membered ring, yielded four stereoisomers (7E,9E,13E, 7E,9E,13Z, 7E,9Z,13E and 7E,9Z,13Z). These four isomers were separated by straight-phase isocratic HPLC and identified by 1H-NMR and NOE analysis. All isomers smoothly recombined with bovine opsin at a relatively high rate (5-10% of that of the natural chromophore 11Z-retinal). The corresponding 13E and 13Z isomers yielded identical analog pigments, probably due to rapid thermal isomerization around the C13 = C14 double bond. The (7E,9E)-isomers produced a pigment with maximal absorbance at 510 nm, while the pigment produced from the (7E,9Z)-isomers had maximal absorbance at 494 nm. Based upon kinetic considerations, the chromophore structure in the 510-nm-absorbing pigment should be (7E,9E,13E), i.e. equivalent to 11Z-retinal and rhodopsin, while the chromophore structure in the 494-nm-absorbing pigment should be (7E,9Z,13Z), i.e. equivalent to (9Z,11Z,13Z)-rhodopsin, an isorhodopsin analog. In analogy to the 11-cis-locked rhodopsin analogs Rh5 and Rh7, the 510-nm-absorbing pigment, (7E,9E,13E)-10,20-methanorhodopsin, was dubbed Rh6 and the 494-nm-absorbing pigment. (7E,9Z,13Z)-10,20-methanorhodopsin, was dubbed Iso6. The opsin shift of Rh6 (2660 cm-1) is practically identical to that of rhodopsin itself (2650 cm-1). Rh6 and Iso6 are nearly as stable as rhodopsin towards hydroxylamine and solubilization in detergent solution and could be easily purified and reconstituted into proteoliposomes by established procedures. Due to the 11-cis-lock, Rh6 is much less photolabile (bleaching rate less than 1%) than rhodopsin, but it is not completely photostable, probably since photoisomerization around the C7 = C8, C9 = C10 and C13 = C14 bonds is allowed. Illumination of either Rh6 or Iso6 does not generate the common photointermediates but instead produces a complex pattern of photochemical transitions, which during continuous illumination leads to the same final steady state, absorbing at 498 nm. This process is accompanied by a slow but steady loss of pigment, probably due to hydrolytic release of chromophore, which is markedly accelerated in the presence of hydroxylamine. In a physiological assay (light-dependent activation of rod cGMP phosphodiesterase) Rh6 is only marginally active and this probably reflects conformational changes accompanying the above-mentioned photochemical transitions. This supports the concept that normal rhodopsin-based phototransduction requires 11Z to all-E isomerization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered olfactory receptor neuron responsiveness in rare Ostrinia nubilalis males attracted to the O. furnacalis pheromone blend

Three percent of E-strain Ostrinia nubilalis males fly upwind in response to the Ostrinia furnacalis pheromone blend [a 40:60 ratio of (E)-12-tetradecenyl acetate to (Z)-12-tetradecenyl acetate (E12-14:OAc to Z12-14:OAc)], in addition to their own pheromone blend [a 99:1 ratio of (E)-11-tetradecenyl acetate to (Z)-11-tetradecenyl acetate) (E11-14:OAc to Z11-14:OAc)]. We assessed the olfactory receptor neuron (ORN) responses of these behaviorally "rare" males versus those of normal males. For the three ORNs housed within each sensillum, we tested responsiveness to Z12-14:OAc, E12-14:OAc, Z11-14:OAc, E11-14:OAc, and the behavioral antagonist (Z)-9-tetradecenyl acetate (Z9-14:OAc). Z11-14:OAc, E11-14:OAc, and Z9-14:OAc stimulated ORNs exhibiting distinct small, large, and medium spike sizes, respectively. For rare and normal males, both Z12-14:OAc and E12-14:OAc usually elicited responses from the largest-spiking ORN. In many ORNs of normal males, Z12-14:OAc or E12-14:OAc stimulated the smaller-spiking ORN that is responsive to Z11-14:OAc. In rare males, detectable ORN responses from the smaller-spiking ORN in response to Z12- and E12-14:OAc were virtually non-existent. These differences in ORN tuning in rare males will tend to create an ORN firing ratio between the large- and small-spiking ORNs in response to the O. furnacalis blend that is similar to that elicited by the O. nubilalis blend.     

Function of redox-active tyrosine in photosystem II

Water oxidation at photosystem II Mn-cluster is mediated by the redox-active tyrosine Y(Z). We calculated the redox potential (E(m)) of Y(Z) and its symmetrical counterpart Y(D), by solving the linearized Poisson-Boltzmann equation. The calculated E(m)(Y( )/Y(-)) were +926 mV/+694 mV for Y(Z)/Y(D) with the Mn-cluster in S2 state. Together with the asymmetric position of the Mn-cluster relative to Y(Z/D), differences in H-bond network between Y(Z) (Y(Z)/D1-His(190)/D1-Asn(298)) and Y(D) (Y(D)/D2-His(189)/D2-Arg(294)/CP47-Glu(364)) are crucial for E(m)(Y(Z/D)). When D1-His(190) is protonated, corresponding to a thermally activated state, the calculated E(m)(Y(Z)) was +1216 mV, which is as high as the E(m) for P(D1/D2). We observed deprotonation at CP43-Arg(357) upon S-state transition, which may suggest its involvement in the proton exit pathway. E(m)(Y(D)) was affected by formation of P(D2)(+) (but not P(D1)(+)) and sensitive to the protonation state of D2-Arg(180). This points to an electrostatic link between Y(D) and P(D2).     

Simple synthesis of mite pheromone beta-acaridial and its analogs in the secretion of Caloglyphus polyphyllae (Acari: Acaridae)

A simple synthesis of beta-acaridial [(E)-1], the active principle of the sex, alarm and aggregation pheromone among astigmatid mites, was achieved in 5 steps from 1,2,4-butanetriol 2 in a 19% overall yield. Its analog, beta-acariolal 8, was also prepared in a 63% yield by oxidation of the intermediate, beta-acaridiol [(E)-7], with pyridinium dichromate (PDC). This synthetic route also gave beta-(Z)-acaridiol [(Z)-7] by using a Z-selective base in the Wittig reaction. (Z)-7 was oxidized to give a new monoterpene, beta-(Z)-acaridial [(Z)-1], which was detected as a trace component in the secretion of Caloglyphus polyphyllae, together with 8.     

Sex pheromone biosynthesis in the pine caterpillar moth, Dendrolimus punctatus (Lepidoptera: Lasiocampidae): pathways leading to Z5-monoene and 5,7-conjugated diene components

Biosynthesis of the sex pheromone components (Z)-5-dodecenol and (Z,E)-5,7-dodecadienol in Dendrolimus punctatus was studied by topical application of deuterium-labeled fatty acids to pheromone glands and subsequent analysis of fatty acyl groups and pheromone components by gas chromatography-mass spectrometry. Our studies suggest that both (Z)-5-dodecenol and (Z,E)-5,7-dodecadienol can be biosynthetically derived from chain elongation of palmitate to stearate in the gland, and its subsequent Delta11 desaturation to produce (Z)-11-octadecenoate. After three cycles of 2-carbon chain-shortening, the pheromone glands produce (Z)-5-dodecenoate, which is then converted to (Z)-5-dodecenol by reduction. A second Delta11 desaturation of (Z)-9-hexadecenoate produces (Z,E)-9,11-hexadecadienoate, which is then chain shortened in two cycles of beta-oxidation and finally converted to (Z,E)-5,7-dodecadienol by reduction.     

Synthesis and structural identification of four dihydroxy acids and 11,12-leukotriene C4 derived from 11,12-leukotriene A4

Using a partially purified 12-lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)-12-hydroperoxy-5,8,10,14-icosate traenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C-12 was determined as S by chiral-phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (11,12-leukotriene A4) and (5Z,7Z,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (7-cis-11,12-leukotriene A4). 11,12-Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)-(12S)-5,12-dihydroxy-6,8,10,14-i cosatetraenoic acid and two isomers of (14Z)-(12S)-11,12-dihydroxy-5,7,9,14-icosatetraenoic acid. Upon incubation with rat liver glutathione S-transferase, 11,12-leukotriene A4 was converted to 11,12-leukotriene C4, a spasmogenic compound.     

Evidence of olfactory antagonistic imposition as a facilitator of evolutionary shifts in pheromone blend usage in Ostrinia spp. (Lepidoptera: Crambidae)

Olfactory receptor neuron (ORN) response was measured to assess why some males ("rare males") of the Asian corn borer (ACB), Ostrinia furnacalis, have a broad behavioral response to fly upwind to both the ACB and the European corn borer (ECB), Ostrinia nubilalis, pheromone blends. We performed single-sensillum electrophysiological recordings on ACB males that had been behaviorally assessed for upwind flight response to the ACB blend [60:40 (Z)-12-tetradecenyl acetate (Z12-14:OAc) to (E)-12-tetradecenyl acetate (E12-14:OAc)], as well as to ECB (Z-strain) and ECB (E-strain) blends [3:97 and 99:1 (Z)-11-tetradecenyl acetate (Z11-14:OAc) to (E)-11-tetradecenyl acetate (E11-14:OAc)]. Sensilla from all types of males had large- and small-spike-sized ORNs responding strongly to Z12- or E12-14:OAc, but weakly to Z11- and E11-14:OAc. In the majority of males ("normal males") that flew upwind only to the ACB blend, Z11-14:OAc elicited responses in an intermediate spike-sized ORN associated with behavioral antagonism that is mainly tuned to (Z)-9-tetradecenyl acetate (Z9-14:OAc). In the rare-type ACB males that flew to both the ACB and ECB pheromone blends, Z11-14:OAc did not stimulate this ORN. Increased responsiveness to ancestral pheromone components by ORNs associated with behavioral antagonism could be instrumental in reproductive character displacement, or in reinforcement and reproductive isolation during speciation by helping to increase assortative mating between males and females in derived populations that use novel sex pheromone blends.     

Antimicrobial activity of the thiosulfinates isolated from oil-macerated garlic extract

Three thiosulfinates were isolated from oil-macerated garlic extract, and their structures were identified as 2-propene-1-sulfinothioic acid S-(Z,E)-1-propenyl ester [AllS(O)SPn-(Z,E)], 2-propenesulfinothioic acid S-methyl ester [AllS(O)SMe], and methanesulfinothioic acid S-(Z,E)-1-propenyl ester [MeS(O)SPn-(Z,E)]. This is the first report of isolating these thiosulfinates from oil-macerated garlic extract. Antimicrobial activities of AllS(O)SPn-(Z,E) and AllS(O)SMe against Gram-positive and negative bacteria and yeasts were compared with 2-propene-1-sulfinothioic acid S-2-propenyl ester [AllS(O)SAll, allicin] which is well-known as the major thiosulfinate in garlic. Antimicrobial activity of AllS(O)SMe and AllS(O)SPn-(Z,E) were comparable and inferior to that of allicin, respectively. This result suggested that the antimicrobial activity of 2-propene sulfinothioic acid S-alk(en)yl esters were affected by alk(en)yl groups. The order for antimicrobial activity was: allyl > or = methyl > propenyl.     

Purification of a lipoxygenase from ungerminated barley. Characterization and product formation

Lipoxygenase was purified from ungerminated barley (variety 'Triumph'), yielding an active enzyme with a pI of 5.2 and a molecular mass of approximately 90 kDa. In addition to the 90 kDa band SDS-PAGE showed the presence of two further proteins of 63 kDa. Western blot analysis showed cross-reactivity of each of these proteins with polyclonal antisera against lipoxygenases from pea as well as from soybean, suggesting a close immunological relationship. The 63 kDa proteins appear to be inactive degradation products of the active 90-kDa enzyme. This barley lipoxygenase converts linoleic acid mainly into (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid, and arachidonic acid into (5S)-(6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eic osatetraenoic acid.     

Behavioural and electrophysiological activity of unsaturated analogues of the pheromone tetradecyl acetate in the small ermine moth Yponomeuta rorellus

ABSTRACT In field-trapping experiments the unsaturated analogues ( E )-6-, ( E )-12-, and ( Z )-12-tetradecenyl acetate were as attractive to male Yponomeuta rorellus Latr. as the native pheromone component tetradecyl acetate. All four analogues attracted more males than virgin females did, whereas ( Z )-6-, ( E )-11-, ( Z )-10- and ( Z )-11-tetradecenyl acetate were essentially non-attractive. Addition of 1–30% of ( Z )-11-tetradecenyl acetate to the pheromone tetradecyl acetate reduced the attraction to less than 2%. Flight tunnel experiments with Y. rorellus confirmed the activity of the ( E )-6- and ( E )-12-tetradecenyl acetates and demonstrated the activity of ( E )-7-tetradecenyl acetate as well. These analogues elicited orientation behaviour, upwind flight and landing at the odour source as frequently as the native pheromone did. Single sensillum recordings from male Y. rorellus showed two types of cells in most sensilla. A large spike amplitude cell was stimulated by tetradecyl acetate and the unsaturated analogues ( E )-11-, ( E )-6- and ( E )-12-tetradecenyl acetate, and to a lower extent by the ( Z )-6-, ( Z )-11- and ( Z )-12-isomers. A cell with medium spike amplitude was stimulated by ( Z )-9-tetradecenyl and ( Z )-11-hexadecenyl acetate. Some sensilla contained a third cell firing with a small spike amplitude which was activated by ( Z )-11-tetradecenol. Thus the tetradecyl acetate receptor was stimulated not only by the behaviourally active analogues, but also by behavioural antagonists. The interaction of ( E )-11-tetra-decenyl acetate and tetradecyl acetate with the same antennal receptor cell was also demonstrated in Y.cagnagellus . Electrophysiological discrimination between behavioural attractants and antagonists and the role of behavioural antagonists in the interspecific relations between Y.rorellus and sympatric closely related species are discussed.     

Flight and molecular modeling study on the response of codling moth, Cydia pomonella (Lepidoptera: Tortricidae) to (E,E)-8,10-dodecadien-1-ol and its geometrical isomers

In a previous study we have reported that both (E,Z)-8,10-dodecadienol (E,Z) and (Z,Z)-8,10-dodecadienol (Z,Z) isomers inhibit the attraction of male codling moth, Cydia pomonella L. when added to (E,E)-8,10-dodecadienol (E,E) while the (Z,E)-8,10-dodecadienol (Z,E) isomer induces slight increase in the number of males attracted to the pheromone source. In the present study, we have tested the behavioral activity of the individual geometrical isomers E,Z; Z,E and Z,Z. A few number of codling moth males flew to the Z,E-isomer while the other two isomers (i.e. E,Z and Z,Z) did not elicit any upwind orientation. Analysis of the flight behavior to the E,E- and Z,E-isomer showed significant differences in most of the flight parameters evaluated. Based on the biological observations and molecular modeling, we suggest that the behavioral activity of the Z,E-isomer is due to presence of specific receptors for this isomer on male antennae and not to its structural resemblance to the E,E-isomer. These results underline the importance of the Z,E-isomer in sex attraction of male codling moth.     

Sex pheromone of Etiella behrii, a pod borer of soybean in Indonesia: identification and field attraction

Four EAG-active components were detected in GC-EAG analyses of hexane extracts from virgin Etiella behrii (Zeller) (Lepidoptera: Pyralidae) females. These components were identified as dodecyl acetate (12:Ac), (E)-9-dodecenyl acetate (E9-12:Ac), either (Z)-9- or (E)-11-tetradecenyl acetate (Z9- or E11-14:Ac), and (Z)-11-tetradecenyl acetate (Z11-14:Ac) by comparison of retention indices on both nonpolar and polar GC columns. The available extract was insufficient for further GC-MS or other chemical analyses (<0.2 ng/female). In field tests carried out in East Java, a 10:90 mixture of E9-12:Ac and Z11-14:Ac showed attractiveness to male moths and addition of 12:Ac and/or E11-14:Ac significantly increased the trap catches while addition of Z9-14:Ac showed no significant effect. Maximum attraction was obtained with 5.35 or 10.7 g/rubber septum of a mixture of E9-12:Ac, Z11-14:Ac, 12:Ac and E11-14:Ac at the ratio of 10:90:0.7:6.3, respectively. The role of pheromone blends in species discrimination between E. behrii and the related E. zinckenella (Treitschke) is discussed.     

Reidentification of the female sex pheromone of the Indian meal moth, Plodia interpunctella: evidence for a four-component pheromone blend

Pheromone gland extracts from calling female Plodia interpunctella contained at least seven compounds that consistently elicited electroantennographic responses from male antennae upon gas chromatographic analysis. Three of these compounds were found to be the previously identified gland constituents, i.e., (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), (Z,E)-9,12-tetradecadienal (Z9,E12-14:Ald) and (Z,E)-9,12-tetradecadienol (Z9,E12-14:OH). A fourth EAD-active compound was identified as (Z)-9-tetradecenyl acetate (Z9-14:OAc). The homologue (Z)-11-hexadecenyl acetate (Z11-16:OAc) was also identified in the extracts, but showed no EAD activity. The identity of all five compounds was confirmed by comparison of GC retention times and mass spectra with those of synthetic standards. In flight tunnel tests there were no significant differences in response of male P. interpunctella to the bait containing all four EAD-active compounds and the responses to female gland extacts. A behavioural assay of different two-compound blends in the flight tunnel showed that only addition of the corresponding aldehyde to the major pheromone component Z9,E12-14:OAc raised the male response. A subtractive assay, however, revealed that the exclusion of any of the compounds from the complete four-compound blend reduced its activity significantly. We thus conclude that the female-produced sex pheromone of P. interpunctella consists of at least four components, i.e., Z9,E12-14:OAc, Z9,E12-14:Ald, Z9,E12-14:OH and Z9-14:OAc.In a field trapping test performed in a storage facility, the four-component blend attracted significantly more males of P. interpunctella than traps baited with Z9,E12-14:OAc alone. In contrast, the highest number of Ephestia kuehniella males was found in the traps baited with this major component, suggesting that the secondary pheromone components contribute to the species specificity of the blend.