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The iridoids and their derivatives monoterpene indole alkaloids (MIAs) are two broad classes of plant-derived natural products with valuable pharmaceutical properties. However, the poor source limited their application. Nepetalactol, a common iridoid scaffold of MIAs, was heterologously produced in Saccharomyces cerevisiae. Although the optimization of nepetalactol production in S. cerevisiae was achieved by metabolic engineering, the inherent metabolic constraints impose a restriction on the production. Herein, we developed a high nepetalactol-producing Aspergillus oryzae platform strain. First, the co-expression of 5 nepetalactol biosynthetic genes, in a high isopentenyl pyrophosphate (IPP)-producing strain A. oryzae AK2, succeeded in the biosynthesis of nepetalactol. Second, the improvement of the IPP supply and the suppression of the byproduct citronellol formation were simultaneously achieved. Finally, the highest titer of nepetalactol of 7.2 mg/L was obtained with the engineered strain, after the optimization of the carbon source. To the best of our knowledge, this is the highest reported titer of nepetalactol in microbial cells. The developed A. oryzae strain represents an attractive biosynthetic platform host for the de novo production of iridoids and MIAs.

Nepetalactol, an iridoid with four chiral carbons, is a crucial component of aphid sex pheromones that have been employed with great success to control the insect-related diseases. Despite of agricultural usage as end products, iridoids are fundamental biosynthetic intermediates for pharmaceutically important monoterpenoid indole alkaloids such as camptothecin (CAM) and vinca alkaloids. Herein we characterized 10-hydroxygeraniol oxidoreductase (10HGO) and iridoid synthase (IS) from Camptotheca acuminata, a CAM-producing plant, and reported their application in biological preparation of nepetalactol. Ca10HGO and CaIS were respectively cloned from C. acuminata, overexpressed in Escherichia coli, and purified to homogeneity. Ca10HGO catalyzes the oxidation of 10-hydroxygeraniol into 10-oxogeranial, in which NADP+ was reduced to NADPH. CaIS catalyzes nepetalactol formation from 10-oxogeranial using NADPH cofactor. The net outcome of the two reactions generate nepetalactol from 10-hydroxygeraniol efficiently, indicating NADP+ - NADPH recycling. Ca10HGO and CaIS were co-overexpressed in E. coli under optimized fermentation conditions to prepare cell-based catalysts that catalyze the conversion of 10-hydroxygeraniol into nepetalactol. The present work shows the enzymatic conversion of 10-hydroxygeraniol into nepetalactol involved in CAM biosynthesis. Co-overexpression of Ca10HGO and CaIS in E. coli is an alternative valuable cell-based biotransformation process with regenerating recycling of NADP+ - NADPH cofactors for nepetalactol preparation.

Cats anoint themselves with plant metabolites that confer mosquito repellence., Domestic cats and other felids rub their faces and heads against catnip (Nepeta cataria) and silver vine (Actinidia polygama) and roll on the ground as a characteristic response. While this response is well known, its biological function and underlying mechanism remain undetermined. Here, we uncover the neurophysiological mechanism and functional outcome of this feline response. We found that the iridoid nepetalactol is the major component of silver vine that elicits this potent response in cats and other felids. Nepetalactol increased plasma β-endorphin levels in cats, while pharmacological inhibition of μ-opioid receptors suppressed the classic rubbing response. Rubbing behavior transfers nepetalactol onto the faces and heads of respondents where it repels the mosquito, Aedes albopictus. Thus, self-anointing behavior helps to protect cats against mosquito bites. The characteristic response of cats to nepetalactol via the μ-opioid system provides an important example of chemical pest defense using plant metabolites in nonhuman mammals.

The attraction range of olfactory response by winged female gynoparae (autumn migrants that give birth to oviparae, the sexual females) and male damson–hop aphids Phorodon humuli (Schrank) is investigated in field experiments over 2 years by analyzing the spatial patterns of catches in concentric circles of yellow-painted traps (60 in total) around a central trap releasing the species' sex pheromone, (1RS,7S,7aS)-nepetalactol. Males are more likely than females to be found in the central trap, with 65.6% of the 1824 males caught there compared with 11.2% of 1346 females. Both morphs are more numerous in traps axial with the mean wind direction and centred on the pheromone-release trap than at other angles. Males are approximately five-fold more numerous in traps downwind than at similar distances upwind of the pheromone, showing that its presence stimulates landing. For males, the estimated active space of the lure extends 6 m downwind. Catches of females are equally numerous up and downwind of the pheromone lure because females orienting on the axis of the pheromone source continue to respond to visual cues in their flight path if they overshoot the olfactory one. For females, the active space of a pheromone lure is less than 2 m downwind. It is unimportant for either morph whether the pheromone-release trap is yellow or transparent. In these experiments, both morphs orient with, track and probably arrive in the pheromone source trap from at least 26 m, the distance to the nearest aphid-infested hops.

Monoterpene indole alkaloids (MIAs) from plants encompass a broad class of structurally complex and medicinally valuable natural products. MIAs are biologically derived from the universal precursor strictosidine. Although the strictosidine biosynthetic pathway has been identified and reconstituted, extensive work is required to optimize production of strictosidine and its precursors in yeast. In this study, we engineered a fully integrated and plasmid-free yeast strain with enhanced production of the monoterpene precursor geraniol. The geraniol biosynthetic pathway was targeted to the mitochondria to protect the GPP pool from consumption by the cytosolic ergosterol pathway. The mitochondrial geraniol producer showed a 6-fold increase in geraniol production compared to cytosolic producing strains. We further engineered the monoterpene-producing strain to synthesize the next intermediates in the strictosidine pathway: 8-hydroxygeraniol and nepetalactol. Integration of geraniol hydroxylase (G8H) from Catharanthus roseus led to essentially quantitative conversion of geraniol to 8-hydroxygeraniol at a titer of 227 mg/L in a fed-batch fermentation. Further introduction of geraniol oxidoreductase (GOR) and iridoid synthase (ISY) from C. roseus and tuning of the relative expression levels resulted in the first de novo nepetalactol production. The strategies developed in this work can facilitate future strain engineering for yeast production of later intermediates in the strictosidine biosynthetic pathway.

Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effects in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis. A class of nepetalactol-related short-chain dehydrogenase/reductases (NEPS) captures a reactive enol intermediate produced by iridoid synthase for cyclization and subsequent oxidation into nepetalactones, the active ingredients in catnip.

Improved syntheses of iridomyrmecin and isoiridomyrmecin, major components of matatabilactone, are described. The synthesis features a direct transformation of nepetalactol into key intermediates by DIBAL-H reduction and provides an expeditious and straightforward production of iridoid lactones for biological investigations of the Matatabi phenomenon.

The monoterpene indole alkaloids (MIAs) are a valuable family of chemicals that include the anticancer drugs vinblastine and vincristine. These compounds are of global significance-appearing on the World Health Organization's list of model essential medicines-but remain exorbitantly priced due to low in planta levels. Chemical synthesis and genetic manipulation of MIA producing plants such as Catharanthus roseus have so far failed to find a solution to this problem. Synthetic biology holds a potential answer, by building the pathway into more tractable organisms such as Saccharomyces cerevisiae. Recent work has taken the first steps in this direction by producing small amounts of the intermediate strictosidine in yeast. In order to help improve on these titers, we aimed to optimize the early biosynthetic steps of the MIA pathway to the metabolite nepetalactol. We combined a number of strategies to create a base strain producing 11.4 mg/L of the precursor geraniol. We also show production of the critical intermediate 10-hydroxygeraniol and demonstrate nepetalactol production in vitro. Lastly we demonstrate that activity of the iridoid synthase toward the intermediates geraniol and 10-hydroxygeraniol results in the synthesis of the nonproductive intermediates citronellol and 10-hydroxycitronellol. This discovery has serious implications for the reconstruction of the MIA in heterologous organisms.

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD(+) , NADPH or NAD(+) /10-oxogeranial were solved. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form the byproduct S-10-oxo-citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer, leading to formation of the cis-enolate, together with rotation about C4-C5, which enables cyclization and lactol production. The structure is similar to that of progesterone-5β-reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for β-face hydride attack in progesterone 5β-reductases and is of general interest in the context of asymmetric synthesis.

Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyses the activation of its substrate 8-oxogeranial into a reactive enol intermediate but does not catalyse the subsequent cyclisation into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) which catalyse the stereoselective cyclisation of the enol intermediate into nepetalactol isomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterisation of the NEPS3 cyclase reveals it binds to NAD+ yet does not utilise it chemically for a non-oxidoreductive formal [4+2] cyclisation. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.

Nepetalactones are accumulated as the main secondary metabolites in the majority of Nepeta species; however, their biosynthetic pathway remains to a great extent unknown, as well as their relationship to the overall physiology of these plants. Nepetalactones most certainly arise through general iridoid biosynthetic pathway leading to nepetalactol, a common precursor of all plant-derived iridoids that is formed in a reaction catalysed by iridoid synthase (IS, EC 1.3.1.99). In this paper, we aimed to localize the sites of nepetalactone biosynthesis and accumulation by comparatively analysing the nepetalactones content and the expression of putative iridoid synthase (NrIS) in leaves of Nepeta rtanjensis Diklic & Milojevic, a trans,cis-nepetalactone and dehydronepetalactone rich species. Another aim was to give an insight into the developmental regulation of nepetalactone biosynthesis in leaves of cultivated N. rtanjensis plants. Knowledge on the spatial-, and developmental- regulation of nepetalactone metabolism could greatly benefit potential growers by enabling them to adequately plan and manage plant cultivation, harvesting, and handling and thus increase yield of bioactive compounds. The expression profile of putative NrIS1 is highly correlated with trans,cis-nepetalactone and dehydronepetalactone content/ratio in leaves of different developmental stages. The results strongly indicate that developmental regulation of nepetalactone biosynthesis in leaves resides largely at the level of gene expression, and suggest an important role of NrIS1. Young leaves exhibited the most intensive nepetalactone biosynthesis and accumulation, but also its conversion to dehydronepetalactone. Thus, the pattern of NL accumulation in leaves can be explained by both the rate of its biosynthesis and metabolic degradation. The gene expression patterns of NrIS1 and NrIS2 transcripts in trichomes and abraded leaves revealed trichomes as the main site of their activity. UHPLC/qqqMS profiling of nepetalactones in trichomes and abraded leaves, complemented with Raman spectroscopy and dichloromethane dipping experiment, supported glandular trichome-specific localization of nepetalactone biosynthesis and accumulation.

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous 'ene'-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.

A over view of the synthesis of enantiopure bioactive molecules such as Nepetalactol isofebrifugine R Montelukast phosphanylamino acids and S Pregabalin are efficiently synthesized from common intermediate as Michael adducts by various types of Michael addition reaction and further Michael adduct are potentially leading towards the synthesis of varieties bioactive molecules

Swertia mussotii is an important medicinal plant found on the Qinghai Tibetan Plateau that has great economic and medicinal value. This plant has enjoyed a long history of use as a curative for hepatitis. The biological activity of secoiridoids, including gentiopicroside and swertiamarin, has been mainly tested for its anti-hepatitis effects. Here, we identify two candidate genes (SmIS1 and SmIS2) that are homologues of iridoid synthase and that are components of the secoiridoid pathway in S. mussotii. Using sequencing and phylogenetic analyses, we confirm that SmIS1 and SmIS2 contain six conserved short-chain dehydrogenases/reductase (SDR) motifs and thus belong to the P5βRs group. The two purified Escherichia coli-expressed proteins reduced 8-oxogeranial to both nepetalactol and iridodials. A comparison of the kinetic parameters of SmIS1 and SmIS2 recombinant proteins revealed that SmIS2 has a lower affinity than SmIS1 for 8-oxogeranial. Transcript levels of the two genes were analysed in three different tissues of S. mussotii using semi-quantitative RT-PCR and RT-qPCR. SmIS1 and SmIS2 expression levels were more abundant in leaves and stems. This investigation adds to our knowledge of P5βRs genes in the secoiridoid synthesis pathway and provides candidate genes for genetically improving S. mussotii by enhancing secondary metabolite production.

This study was performed to investigate the response of the lacewing Chrysopa cognata to both Aphis gossypii-induced plant volatiles and lacewing-derived volatiles. The results of a Y-tube olfactometer bioassay showed that more C. cognata males were attracted to green pepper plants infected with A. gossypii than to uninfected green pepper plants alone or clean air and C. cognata males were attractive to C. cognata females. Gas chromatography-electroantennographic detection (GC-EAD) analysis showed that the antennae of C. cognata females elicited EAD-active responses to the volatiles entrained from A. gossypii-infected green pepper plants. 4-Ethylacetophenone, 3-ethylbenzaldehyde, 3-ethylacetophenone, and 4-ethylbenzaldehyde from A. gossypii-induced green pepper volatiles, and (Z,Z)-4,7-tridecadiene, (Z)-4-tridecene, and (Z)-4-undecene from C. cognata female entrainment were elucidated by further analysis using GC coupled nuclear magnetic resonance spectroscopy. Of the A. gossypii-induced plant volatiles identified in this study, 4-ethylacetophenone and 3-ethylbenzaldehyde significantly increased the attraction of C. cognata males to nepetalactol, but (Z)-4-tridecene and (Z)-4-undecene did not. (Z,Z)-4,7-Tridecadiene significantly reduced the attractiveness of nepetalactol to C. cognata.

The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities. Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies. To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels-Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural and pharmaceutical applications.

Green lacewings (Neuroptera: Chrysopidae) are voracious predators of aphids and other small, soft-bodied insects and mites. Earlier, we identified the first lacewing pheromone from field-collected males of the goldeneyed lacewing, Chrysopa oculata Say; (1R,2S,5R,8R)-iridodial is released from thousands of microscopic dermal glands on the abdominal sternum of males, along with comparable amounts of nonanal, nonanol and nonanoic acid. Iridodial-baited traps attract C. oculata and other Chrysopa spp. males into traps, while females come to the vicinity of, but do not usually enter baited traps. Despite their healthy appearance, normal fertility and usual amounts of C9 compounds, laboratory-reared C. oculata males do not produce iridodial. However, we observed that goldeneyed lacewing males caught alive in iridodial-baited traps sometimes try to eat the lure, and in Asia Chrysopa spp. males reportedly eat the native plant, Actinidia polygama (Siebold & Zucc.) Maxim. (Actinidiaceae) to obtain the iridoid, neomatatabiol. These observations prompted us to investigate why laboratory-reared Chrysopa green lacewings do not produce iridodial. Lacewing adult males fed various monoterpenes reduced carbonyls to alcohols and saturated double bonds, but did not convert these compounds to iridodial. Males fed the bicyclic iridoid aphid pheromone component, (4aS,7S,7aR)-nepetalactone, converted ~75% to dihydronepetalactone, but did not produce iridodial; however, wild C. oculata males collected in May often contained traces of dihydronepetalactone. On the other hand, adult males fed the second common aphid pheromone component, (1R,4aS,7S,7aR)-nepetalactol, converted this compound to iridodial. In California the peak late-season attraction of green lacewings to nepetalactol (the lactone is unattractive) occurs at least a month earlier than the peak in aphid oviparae (the pheromone producing morph of aphids), consistent with the hypothesis that Chrysopa males feed on oviparae to obtain nepetalactol as a precursor to iridodial. Adult males from laboratory-reared C. oculata larvae fed nepetalactol failed to produce iridodial, and wild C. oculata males collected early in the spring produce less iridodial than males collected later in the season. Therefore, we further hypothesize that Asian Chrysopa eat A. polygama to obtain iridoid precursors in order to make their pheromone, and that other iridoid-producing plants elsewhere in the world must be similarly usurped by male Chrysopa species to sequester pheromone precursors. Whether or not sequestration of iridodial precursors from oviparae and/or iridoid-containing plants is truly the explanation for lack of pheromone in laboratory-reared Chrysopa awaits further research .

Studies regarding pheromes consisting of essential oil components of various plants and released by insects and arachnids (pests) were compiled for this review. For example, verbenol and verbenone were found to be an essential part of the bark beetle's aggregation pheromone, released with the intent to round up its conspecifics. Concerning plant lice, aphids use (E)-β-farnesene as an alarm pheromone. Furthermore, nepetalactol and nepetalactone, two essential oil components of the cat mint (Lamiaceae), occur as an aphid-released sex pheromone, emitted to induce mating behaviour. On the other hand, some bees imitate a linalool-containing orchid odour as their sex pheromone and thus attract their mating partners. The sand fly, responsible for the American visceral leishmaniasis, uses derivatives of either α-himachalene, germacrene D or cembrene as a sex pheromone. Studies about termites reveal a large amount of different presumable pheromone compounds that also appear as essential oil components, e.g. neocembrene as a part of the trail pheromone, (E,E)-α-farnesene as an alarm pheromone and other terpenes, such as for example γ-cadinene as primer pheromone, enforcing the effect of the juvenile hormone and therefore determining about caste development. Concerning the pest insects, management strategies, conducted with pheromone traps, are presented or compared. Regarding arachnids, studies about spider pheromones are rare, whereas more information is available about mites, which release neral and geranial as an alarm pheromone. Copyright © 2011 John Wiley & Sons, Ltd.

The ant-associated iridoids nepetalactol, actinidine, dolichodial, isoiridomyrmecin, and dihydronepetalactone were prepared from citronellal using a divergent approach. Key features include a three-step synthesis of the individual antipodes of actinidine by a novel tandem cycloaddition/pyridine formation and a facile diastereoselective synthesis of both enantiomers of dolichodial.

An asymmetric synthetic route to cis,cis-nepetalactol (component of the sex pheromone for the hop aphid, Phorodon humuli) is presented. 2-Phenylindoline was resolved to provide a chiral auxiliary for the cycloaddition of oxocitral. The resolution was made by chromatographic separation of the diastereomers of the urea derivative made from 2-phenylindoline and with (R)-(+)-alpha-methylbenzyl isocyanate, followed by reductive cleavage of the isolated diasteromers using diborane. The cycloaddition of oxocitral using (S)-2-phenylindoline yielded an enantiopure product after chromatography. Hydrolysis of the cycloaddition adduct yielded gastrolactol (3). As gastrolactol is a versatile synthon for the synthesis of iridoids, the overall procedure provides a general asymmetric route to elaborated iridoids.

SUMMARY Insect communication is primarily via chemicals. In Aphidinae aphids, the structure and ratio of iridoid (monoterpenoid) chemicals are known to be important components of the sex pheromone. However, for enhanced species specificity, it has been suggested that release of sex pheromone might be restricted to a narrow time period within the diel cycle. Here, we determine the structure, ratios and release patterns of iridoid chemicals produced by a serious global pest, the rosy apple aphid, Dysaphis plantaginea. Volatiles were collected from batches of oviparae (sexual females) and chemicals identified by gas chromatography, mass-spectrometry and microscale NMR spectroscopy.(1R,4aS,7S,7aR)-Nepetalactol and(4aS,7S,7aR)-nepetalactone were detected in a 3.7:1 ratio. To investigate timing of release, we constructed a sequential sampling device that allowed volatile chemicals to be captured hourly from 95 same-aged oviparae over 20 consecutive days. Release patterns of the two sex pheromone components show that D. plantaginea oviparae release high levels of the two components during photophase and low levels during scotophase. Release of the two components increased significantly during the first 3 h of photophase and thereafter remained at a high level until the onset of scotophase. The ratio of(1R,4aS,7S,7aR)-nepetalactol to(4aS,7S,7aR)-nepetalactone released did not change significantly between days two to 14 of the adult stadium, but from the 15th day onward there was a significant decrease in the relative amount of(1R,4aS,7S,7aR)-nepetalactol. Pheromone release was greatest on the eighth day of the adult stadium, with up to 8.4 ng of pheromone released per ovipara per hour. This is the first report on the full structural identification and ratios of volatile iridoid components collected from D. plantaginea oviparae and is also the most detailed temporal study on sex pheromone release from any aphid species. The lack of a temporally narrow and distinct period of very high sex pheromone release suggests that alternative mechanisms or factors for species recognition and isolation may be important. Findings are discussed broadly in relation to the biology of the aphid.

Synthetic (1R,2S,5R,8R)-iridodial, the key pheromone component of many green lacewings in the genus Chrysopa, strongly attracted adult males and females of the North American antlion, Dendroleon speciosus Banks. In addition, one of the common sex pheromone components of many aphids to which Chrysopa spp. are weakly attracted, Z,E-nepetalactol, was also weakly attractive to D. speciosus adults. Iridodial and Z,E-nepetalactol also elicited strong and weak electroantennogram detector (EAD) responses, respectively, in D. speciosus adults. Previously reported semiochemicals from European and Israeli antlion species did not elicit EAD or behavioral responses from D. speciosus adults. The earlier studied antlions release volatile chemicals from male-specific metathoracic glands associated with structures on the hind wings (Eltringham’s organs) that are thought to enhance evaporation of the secretion. Although D. speciosus males have Eltringham’s organs similar to those of other antlion species, we discovered that D. speciosus males have a pair of white tubular glands that extend posteriorly into the abdomen, opening in the resting pits of the Eltringham’s organs. Further gas chromatograph (GC)-EAD analysis of another commercially available antlion species, Myrmeleon crudelis Walker, showed that this species did not respond to the lacewing or aphid related volatile compounds, but strongly responded to the reported antlion semiochemicals, namely, nerol, 10-homonerol and nerol oxide. The male-specific abdominal glands of D. speciosus are presumably pheromone glands; hopefully, positive verification of this supposition will help clarify the semiochemical relationships between antlions, lacewings and aphids.

Gas chromatographic-electroantennographic detection (GC-EAD) analysis showed that male antennae of the green lacewing, Chrysopa nigricornis Burmeister, the most common lacewing species in the U.S. Pacific Northwest, consistently responded to two compounds in thoracic extracts of conspecific males: 1-tridecene and (1R,2S,5R,8R)-iridodial. These compounds were not detected in extracts of the abdominal cuticle, and no other antennally active compounds were found in the abdominal samples. In field-trapping experiments, traps baited with iridodial significantly attracted large numbers of C. nigricornis males (both western and eastern forms) during summer and early fall, plus a few individuals of conspecific females only in early fall. Iridodial also attracted males of the goldeneyed lacewing, C. oculata Say, and, to a lesser extent, C. coloradensis Banks males. Methyl salicylate (MS), reported as an attractant for both sexes of C. nigricornis and C. oculata, was inactive by itself at the concentration tested in our study, but in a few instances slightly enhanced the responses of Chrysopa spp. to iridodial. However, MS alone and its binary blend with iridodial seemed to attract the hoverfly, Metasyrphus americanus (Weidemann). 2-Phenylethanol, a reported attractant for another lacewing, Chrysoperla plorabunda (Fitch) [=carnea (Say)], did not capture any lacewings. Our assays indicated that the lacewing pheromone, iridodial, loaded onto either rubber septa or as a binary blend with MS in polyethylene bags could last at least 5 wk in the field during the summer season. Based on this study, a new attractant system for green lacewings is being developed for both domestic and international markets.

Aphis spiraecola (Patch) virginiparae were reared for 3 generations on the dropwort, Oenathe javanica DC, under low temperature (15°C) and short days (10L:14D) for artificial induction of sexual forms (Hong and Boo, 1998). These oviparae, artificially induced in the third generation in the laboratory, showed a typical behavior of releasing sex pheromone by waving their hind tibiae and abdomen in the air during photophase. The air around these oviparae, individually or in groups, was collected and analyzed by gas chromatography for pheromone components. (+)-(4aS,7S,7aR)-nepetalactone and (-)-(1R,4aS,7S,7aR)- nepetalactol were detected in the ratio of 6:1 ∼ 8:1. For wild oviparae collected from an apple orchard and a spiraea arboretum during Oct. 1995. ∼ Nov. 1996, the ratio between the two components was around 2:1. Oviparae showed a circadian rhythm in release of sex pheromone, with beginning to release nepetalactone at the start of lights-on and stopping with lights-off. This rhythm was also maintained in an all dark condition. The sex pheromone was always detected during the photophase, even when the photoperiod was altered to a longer regime, for example from 10L:14D to 16L:8D.

Gynoparous female and male damson-hop aphids, Phorodon humuli (Schrank), were caught in the field by water traps that were releasing the sex pheromone of this species, (1RS,4aR,7S,7aS)-nepetalactol. No behavioral activity was elicited by (4aS,7S,7aR)-nepetalactone, the major sex pheromone component of other aphid species such as Megoura viciae Buckton, even though olfactory cells were found in the secondary rhinaria on the third antennal segment of P. humuli that responded strongly to this compound. Gynoparous female P. humuli in the field responded less strongly to (1R,4aS,7S,7aR)-nepetalactol, the sex pheromone of the bird cherry-oat aphid, Rhopalosiphum padi (L.), than they did to the (4aR,7S,7aS)-nepetalactols, but males responded only to the latter. The (4aR,7S,7aS)-nepetalactone showed no electrophysiological activity so was not used in field trials. Releasing either the (4aS,7S,7aR)-nepetalactone or the (1R,4aS,7S,7aR)-nepetalactol with the (4aR,7S,7aS)-nepetalactols did not inhibit the response of P. humuli gynoparous females and males to the latter. Males of R. padi responded as strongly to the (4aR,7S,7aS)-nepetalactols as they did to (1R,4aS,7S,7aR)-nepetalactol. Males of P. humuli and R. padi responded positively to an increased concentration of the (4aR,7S,7aS)-nepetalactols released from two vials compared with that from a single vial, as did P. humuli (in one of two experiments) and R. padi to the (1RS,4aR,7S,7aS)- and (1R,4aS,7S,7aR)-nepetalactols when released together.

Electroantennogram (EAG) responses to the aphid sex pheromone components, (1R,4aS,7S,7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone, and a plant volatile, (E)-2-hexenal, were investigated at three different positions (5/6th, 4/5th and 3/4th inter-segmental regions) along the antennae of four different morphs in two host-alternating aphid species, Aphis fabae Scopoli and Rhopalosiphum padi (L.). Position-dependent and morph-specific EAG responses were elicited in both species. The nepetalactol and nepetalactone isomers elicited large EAG responses at all three recording positions in males of both species, such that primary rhinaria as well as secondary rhinaria appeared to respond. Asexual female morphs showed relatively smaller EAG responses to these compounds. The secondary rhinaria, which have been reported as sex pheromone receptors in males, were not very different in their number and distribution between gynoparae and alate virginoparae, but the gynoparae showed significantly larger EAG responses to nepetalactol and nepetalactone. The alate virginoparae showed EAG responses that were similar to those of apterous virginoparae, which lack secondary rhinaria. Taking the EAG response profiles together with the distribution of the secondary rhinaria, it is suggested that the function of secondary rhinaria differs between the morphs. Secondary rhinaria appear to detect sex pheromone components in males and gynoparae but not in the alate virginoparae. If they are functional in the latter morph, they are likely to play a role in detecting specific, but as yet unknown, volatile compounds. Some 30 plant volatiles were tested but none evoked an EAG response that could be allocated to the secondary rhinaria. In contrast to the very different EAG response profiles to the pheromone compounds between morphs, EAG responses to (E)-2-hexenal were similar in all forms and both species. These findings suggest that this plant volatile was detected only by the two primary rhinaria, which are common to all morphs. The present study showed that EAG responses were not a simple summation of receptor potentials between recording and reference electrodes in aphids. The localised distribution pattern of olfactory receptor neurones around the recording electrode was also likely to contribute to the EAG responses.

Outdoor trials were made to test the responses of male aphids to pheromone lures in fields with different host-plants, in order to know whether host-plants would affect catches of males to pheromone lures. In peach orchard, males of three aphid species, i.e. Myzus persicae, Hyalopterus amygdali and Rhopalosiphum nymphaeae, were found in traps baited with aphid sex pheromone nepetalactone and nepetalactol. The most numerous numbers was M, persicae and it was also attracted by plant-derived nepetalactone alone in peach orchard and cabbage garden. Schizaphis graminum males were caught in water traps in wheat field. In apple orchard, males of two aphid species, Aphis cifricola and Sappaphis sinipiricola were caught in significantly larger numbers in the pheromone traps than in the control traps. Field study indicated that the sex pheromone of H. amygduli comprises either one or both of nepetalactone and nepetalactol, and nepetalactone may be one component of sex pheromones of A cifricola and S sinipiricola Another field trial had shown that catches were increased if volatiles from a steam-distilled extract of peach leaves were released simultaneously with nepetalactone. Elec-troantennograms (EAGs) were recorded from males of M. persicae to volatiles of peach leaves and buds.

Field trials by sex pheromone of aphid to trap peach aphids Myzus persicae have been carried out in 1995 and in 1996. Suitable time and the effect of ratio of two components nepetalactone and nepetalactol to apply the lure have been observed.

Male blackberry-cereal aphids, Sitobion fragariae (Walker), were caught in the ield by water traps releasing synthetic (4aS, 7S, 7aR)-nepetalactone, the major sex pheromone component. The presence of the enantiomer (4aR,7R,7aS)-nepetalactone reduced catches and a plant-extracted (4aS,7S,7aR)-nepetalactone was less effective than the 99% (7S)-lactone. A more extensive trial with the bird cherry-oat aphid, Rhopalosiphum padi, involving the pheromone comprising the stereochemically related (4aS,7S,7aR)-nepetalactol showed a similar trend. The (7R)-isomer caught fewer males than the (7S)-isomer, but in this case the addition of high-purity (7S)-lactol to make a 50% blend caught as many males as pure (7S)-lactol. With plant-derived lactol, further purification did not significantly increase the catch. It is suggested that trace compounds associated with reduced enantiomeric purity in terms of the (7S)-configuration or from plant sources reduce activity of the sex pheromone components. Male damson-hop aphids, Phorodon humuli (Schrank), were also caught in the synthetic lactol traps, and it is suggested that this is due to traces of the (4aR,7S,7aS)-nepetalactols, which comprise the sex pheromone for this species. The significance for aphid chemical ecology studies and pest control strategies is discussed.

Field experiments employing yellow water-traps with vials releasing methyl salicylate, butyl isothiocyanate, 4-pentenyl isothiocyanate and diethyltoluamide were conducted during the spring migration of Phorodon humuli (Schrank), with the aim of identifying substances which might be used in the field to deter landing on hop plants. Methyl salicylate and the two isothiocyanates reduced trap catches of P. humuli. During the spring of 1994 a slow-release formulation of methyl salicylate and a beta-acid-rich hop resin sprayed on to hop plants did not reduce aphid infestations significantly. In autumn cis,cis-nepetalactol the main component of P. humuli's sex pheromone, prepared by various synthetic routes, increased trap catches of males and gynoparae equally. Catches of males in pheromone traps situated in a hop garden decreased with increasing trap height. Catches of males in traps charged with increasing doses of the cis,cis-nepetalactol peaked at 1 mg and then plateaued, whereas catches of gynoparae peaked similarly at 1 mg and then decreased. The effects of kairomones from an extract of the primary host, sex pheromone and a visual cue from yellow compared with clear water-traps were additive. The prospects for developing a semiochemicals-based control strategy against P. humuli, using some or all of the above elements, are discussed.

Electroantennograms (EAGs) were recorded from adult male and asexual forms (winged and wingless virginoparae and gynoparae) of the black bean aphid, Aphis fabae, during stimulation with two sex pheromone components, (+)-(4aS,7S,7aR)-nepetalactone and (-)-( lR,4aS,7S,7aR)-nepetalactol, as well as six plant volatiles, i.e. (E)-Zhexenal, (E)-2-hexenol-l, (Z)-3-hexenol-l, (Z)-3-hexenyl acetate, hexanal and ally1 isothiocyanate. The male antennae are 1OOO- 10,OOO times more sensitive to nepetalactol and nepetalactone than to the plant compound (E)-2-hexenal. Besides this marked difference of EAG peak responses in males, the EAG rise and decay are slower for the sex pheromone components. Males are also much more sensitive to the sex pheromone components than asexual females. This high sensitivity correlates with a predominance of antenna1 secondary rhinaria, the major sites of phero- mone perception in the male. However, it is the primary rhinaria on the antennae of the wingless asexual females that are responsible for pheromone perception. Male antennae are as responsive as the asexual female antennae to the plant volatiles. The specialization of the male for mate location is discussed.

The sex pheromone of the grain aphid,Sitobion avenae (Fab.), is identified as (4aS,7S,7aR)-nepetalactone, for which specific olfactory cells are located in the secondary rhinaria of the male antenna. The related (1R,4aS,7S,7aR)-nepetalactol was not produced by females in significant amounts (ca 10,000-fold less than the nepetalactone), although cells in the male antenna were found that responded specifically to this compound. Behavioural activity in the linear track ol-factometer showed attraction of males to the nepetalactone, but not to the nepetalactol.

Mealy plum, Hyalopterus pruni, and leaf-curl plum, Brachycaudus helichrysi, aphids are the primary arthropod pests in orchards that produce dried plums (i.e., prunes). The sexual stage of their respective lifecycles occurs on prune trees in the fall, during which time males respond to sex pheromones produced by oviparous females. Air-entrainment collections confirmed that oviparous H. pruni and B. helichrysi emitted combinations of (4aS, 7S, 7aR)-nepetalactone and (1R, 4aS, 7S, 7aR)-nepetalactol. The responses of H. pruni and B. helichrysi to these compounds in ratios of 1:0, 0:1, 1:1, 2.6:1, 3.4:1, 5:1, 7:1, and 0:0 (no-pheromone control) using water traps were determined in field experiments conducted in prune orchards during the fall. The greatest number of male H. pruni was caught in traps releasing a 1:1 ratio of (4aS, 7S, 7aR)-nepetalactone and (1R, 4aS, 7S, 7aR)-nepetalactol, while male B. helichrysi were caught in similar numbers in traps releasing any of the two-component ratios tested. There was no evidence that any of the pheromone treatments influenced trap catches of gynoparae of either species. Results suggest that addition of sex pheromone lures increases trap catches of male H. pruni and B. helichrysi, and that this approach may improve monitoring and management of these pests in prune orchards. Knowledge gained from this study contributes to the understanding of the ecology of insect pests in prune orchards.

Transparent plastic water traps, baited with the synthetic aphid sex pheromone components (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepetalactol or control lures, were placed in four semi-sheltered field sites at a height of 1.1 m. Although males of twenty-one aphid species were collected from the water traps, onlySitobion fragariae (Walker) was abundant. In total, 8 pheromone traps produced 102 males compared with only 10 males in 8 control traps. The sex pheromone released by sexual femaleS. fragariae was identified as the nepetalactone used in the lures. Aphid sex pheromones may be more species-specific than previously thought and the presence of a host plant is not essential for males to locate sexual females.